Gene composition for detecting cell proliferative abnormality or grading disease degree and use thereof

ABSTRACT

The present invention provides a composition, a kit and the use thereof, as well as the method for detecting the cell proliferative abnormality in individuals or grading the disease degree in the individuals. The composition comprises nucleic acids for detecting the methylation level within at least one target region of a gene and the fragment thereof.

This application claims the priority of a patent application for aninvention under the application No. 2014103898952, filed on Aug. 8,2014, entitled “gene composition for detecting cells proliferativeabnormality or grading disease degree and use thereof”.

TECHNICAL FIELD

The present invention relates to gene detection, particularly relates toa gene composition and use of the gene composition for detectingabnormal cell proliferation in an individual or grading diseaseseverity.

BACKGROUND ART

The disease caused by abnormal cell proliferation, such as cancer, is amajor challenge for public health. In the United States, the death tolldue to cancers accounts for about 25% of the total death toll. Amongthese, gastric cancer, as the fourth cancer most commonly diagnosed inthe world with the second mortality ranking in all cancers, isconsidered as one of the largest enemies to health worldwide. Especiallyin many Asian countries including in China, gastric cancer ranks firstin various malignancies, with very high morbidity and mortality.

Gastritis is a general name for the inflammation in gastric mucosa,which is common in adults. Many etiological factors, such as improperdiets, viral and bacterial infections, drug stimulation, can stimulatestomach and cause gastritis, but gastritis is relatively easy to becorrected and treated.

However, it is often easy to confuse the symptoms of gastritis andgastric cancer, so that many people often mistake gastritis for gastriccancer in daily life and fear all day long. Even if gastritis is not aserious disease, it will continue to deteriorate due to psychologicaleffects. Meanwhile, some people mistake gastric cancer for gastritis andoverlook it, and believe that it is not a serious problem. As a result,they miss the best time for treatment.

Therefore, there is an urgent need for a detection method with highsensitivity and specificity so as to distinguish among normal, gastritisand gastric cancer, in particular for a detection method with highsensitivity and specificity without pain.

With the continuous development of biotechnology, use of gene detectionin diagnosis method for diseases has attracted extensive attentions.Among these, DNA methylation is an important component of epigenetics,which plays an important role not only in the maintenance of normal cellfunctions, but also in the occurrence of cancer and inflammation, thatis, the change of methylation status is an important factor in theoccurrence of cancer and inflammation, and includes decreasedmethylation level of whole genome and abnormally increased localmethylation level of CpG island, leading to instable genome andexpression failure of disease-resistant genes. If an active allele of adisease-resistant gene is inactivated, the risk of the occurrence ofcancer and inflammation will increase. Therefore, a research onmethylation provides a new basis for the early prediction,classification, grading and prognosis evaluation of cancer andinflammation, and becomes one of the current research focuses.

Contents of Invention

Therefore, the present invention provides a gene composition and a kitfor detecting abnormal cell proliferation in an individual or gradingdisease severity (such as gastric cancer and gastritis) by detecting themethylation level, and use thereof, as well as a method for performingthe detection based on the kit.

Accordingly, according to the first aspect of the present invention,provided is a composition comprising a nucleic acid for detecting themethylation level within at least one target region of RNF180 andSeptin9 genes, or fragments thereof.

Typically, the nucleic acid comprises a long fragment of at least 15oligonucleotides of RNF180, wherein the oligonucleotides comprise atleast one CpG dinucleotide sequence.

According to the certain preferred embodiments, the long nucleotidefragment of RNF180 comprises at least 15 nucleotides which areequivalent to, complementary to, or hybridize under moderately stringentor stringent conditions to a sequence selected from the group consistingof SEQ ID Nos: 10 to 12, or complementary sequence thereof.

Further preferably, the long nucleotide fragment of RNF180 comprises asequence which is equivalent to, complementary to, or hybridizes undermoderately stringent or stringent conditions to a sequence selected fromthe group consisting of SEQ ID Nos: 1 to 9, or complementary sequencethereof.

Typically, the composition further comprises a reagent that converts5-unmethylated cytosine base of a gene to uracil or other base that isdetectably different from cytosine in terms of hybridizationperformance. Preferably, the reagent is bisulfite.

According to the second aspect of the present invention, furtherprovided is a kit comprising the composition described above.

Typically, the kit comprises a container for containing a biologicalsample from a patient.

Also, the kit may also comprise the instructions for using the kit andexplaining the results of the kit.

According to the third aspect of the present invention, further providedis use of the composition in the manufacture of a kit for detectingabnormal cell proliferation in an individual.

Typically, the abnormal cell proliferation is a cancer. Preferably, thecancer is gastric cancer.

According to the fourth aspect of the present invention, furtherprovided is a method for detecting abnormal cell proliferation in anindividual, comprising determining the methylation level within at leastone target region of RNF180 and Septin9 genes or fragments thereof in abiological sample isolated from the individual, and detecting abnormalcell proliferation in the individual by combining the detection resultsof methylation of RNF180 and Septin9.

Typically, the method further comprises: treating Septin9 and RNF180genes or fragments thereof with a reagent which convert 5-unmethylatedcytosine base of a gene to uracil or other base that is detectablydifferent from cytosine in terms of hybridization performance;contacting Septin9 and RNF180 genes or fragments thereof treated by thereagent with an amplification enzyme and primers such that the treatedgenes or fragments are amplified to produce amplification products ornot amplified; detecting the amplification products with probes; anddetermining the methylation level of at least one CpG dinucleotide ofthe DNA sequences of Septin9 and RNF180 genes based on the presence orabsence of the amplification products.

Typically, the primers comprise a long nucleotide fragment of RNF180comprising at least 15 nucleotides which are equivalent to,complementary to, or hybridize under moderately stringent or stringentconditions to a sequence selected from the group consisting of SEQ IDNos: 10 to 12, or complementary sequence thereof.

Preferably, the long nucleotide fragment of RNF180 comprises a sequencewhich is equivalent to, complementary to, or hybridizes under moderatelystringent or stringent conditions to a sequence selected from the groupconsisting of SEQ ID Nos: 1 to 9, and complementary sequence thereof.

Wherein, the primers and probes are preferably screened with anartificially methylated template and an unmethylated template; or theprimers and probes are screened with cancer and normal DNA as templates.

Typically, the biological sample of an individual is selected from thegroup consisting of cell lines, histological sections, tissuebiopsies/paraffin-embedded tissues, body fluids, stool, coloniceffluent, urine, plasma, serum, whole blood, isolated blood cells, cellsisolated from blood, or combination thereof.

Preferably, the biological sample of an individual is plasma.

According to certain preferred embodiments, the methylation status of atleast one CpG dinucleotide of the DNA sequences of Septin9 and RNF180gene is determined by the cycle threshold Ct value of a polymerase chainreaction.

Typically, the abnormal cell proliferation is a cancer. Preferably, thecancer is gastric cancer.

Accordingly, according to another aspect of the present invention,provided is use of a composition in the manufacture of a kit for gradingdisease severity in an individual. The composition comprises a nucleicacid for detecting the methylation level within at least one targetregion of RNF180 gene or fragment thereof, and the disease severity isgraded by the detection result of the methylation level of RNF180.

According to certain preferred embodiments of the present invention, thecomposition further comprises a nucleic acid for detecting themethylation level within at least one target region of Septin9 gene orfragment thereof, and the disease severity is graded by combining thedetection results of methylation of RNF180 and Septin9.

Typically, the nucleic acid comprises a long fragment of at least 15oligonucleotides of RNF180, wherein the oligonucleotides comprise atleast one CpG dinucleotide sequence.

Preferably, the nucleotide long fragment of RNF180 comprises at least 15nucleotides which are equivalent to, complementary to, or hybridizeunder moderately stringent or stringent conditions to a sequenceselected from the group consisting of SEQ ID Nos: 10 to 12, orcomplementary sequence thereof.

Further preferably, the nucleotide long fragment of RNF180 comprises asequence which is equivalent to, complementary to, or hybridizes undermoderately stringent or stringent conditions to a sequence selected fromthe group consisting of SEQ ID Nos: 1 to 9, or complementary sequencethereof.

Typically, the composition further comprises a reagent that converts5-unmethylated cytosine base of a gene to uracil or other base that isdetectably different from cytosine in terms of hybridizationperformance. Preferably, the reagent is bisulfite.

Typically, the disease severity is divided into three levels, and thefirst level is normal, the second level is inflammation and the thirdlevel is cancer. In certain preferred embodiments, the inflammation isgastritis, and the cancer is gastric cancer. Preferably, the gastritiscan be further graded into the gastritis that is less prone tocanceration and the gastritis that is prone to canceration. Thegastritis that is less prone to canceration is superficial gastritis.Further preferably, the gastritis that is prone to canceration isfurther graded into atrophic gastritis and gastritis with intestinalmetaplasia. Further preferably, gastric cancer may be further gradedinto gastric cancer stages I, II, III and IV.

According to another aspect of the present invention, provided is amethod for grading disease severity in an individual, comprisingdetermining the methylation level within at least one target region ofRNF180 or fragment thereof in a biological sample isolated from theindividual, and grading the disease severity by the detection result ofmethylation of RNF180.

Typically, the method further comprises determining the methylationlevel within at least one target region of Septin9 gene or fragmentthereof in a biological sample isolated from the individual, and gradingthe disease severity by combining the detection results of methylationof RNF180 and Septin9.

According to certain preferred embodiments, the method furthercomprises: treating Septin9 and RNF180 genes or fragments thereof with areagent which converts 5-unmethylated cytosine base of a gene to uracilor other base that is detectably different from cytosine in terms ofhybridization performance; contacting Septin9 and RNF180 genes orfragments thereof treated by the reagent with an amplification enzymeand primers such that the treated genes or fragments are amplified toproduce amplification products or not amplified; detecting theamplification products with probes; and determining the methylationlevel of at least one CpG dinucleotide of the DNA sequences of Septin9and RNF180 gene based on the presence or absence of the amplificationproducts.

Typically, the primers comprises a nucleotide long fragment of RNF180comprising at least 15 nucleotides which are equivalent to,complementary to, or hybridize under moderately stringent or stringentconditions to a sequence selected from the group consisting of SEQ IDNos: 10 to 12, or complementary sequence thereof.

Preferably, the nucleotide long fragment of RNF180 comprises a sequencewhich is equivalent to, complementary to, or hybridizes under moderatelystringent or stringent conditions to a sequence selected from the groupconsisting of SEQ ID Nos: 1 to 9, or complementary sequence thereof.

Wherein, the primers and probes are preferably screened with anartificially methylated template and an unmethylated template; or theprimer and probes are screened with cancer and normal DNA as templates.

Typically, the biological sample of an individual is selected from thegroup consisting of cell lines, histological sections, tissuebiopsies/paraffin-embedded tissues, body fluids, stool, coloniceffluent, urine, plasma, serum, whole blood, isolated blood cells, cellsisolated from blood, or combination thereof.

Preferably, the biological sample of an individual is plasma.

According to certain preferred embodiments, the methylation status of atleast one CpG dinucleotide of the DNA sequences of Septin9 and RNF180genes is determined by the cycle threshold Ct value of a polymerasechain reaction.

Typically, the disease severity is divided into three levels, and thefirst level is normal, the second level is inflammation and the thirdlevel is cancer.

According to another aspect of the present invention, further providedis a nucleic acid comprising at least 15 nucleotides which areequivalent to, complementary to, or hybridize under moderately stringentor stringent conditions to a sequence selected from the group consistingof SEQ ID Nos: 10 to 12, or complementary sequence thereof.

Preferably, the nucleic acid comprises a sequence which is equivalentto, complementary to, or hybridizes under moderately stringent orstringent conditions to a sequence selected from the group consisting ofSEQ ID Nos: 1 to 9, or complementary sequence thereof.

In certain preferred embodiments, the nucleic acid is used as a primerand/or a probe for detecting the methylation level within at least onetarget region of RNF180 gene or fragment thereof.

According to another aspect of the present invention, further providedis a composition comprising the nucleic acid, further a reagent thatconverts 5-unmethylated cytosine base of a gene to uracil or other basethat is detectably different from cytosine in terms of hybridizationperformance. Preferably, the reagent is bisulfite.

According to another aspect of the present invention, further providedis a kit comprising the composition described above. Typically, the kitcomprises a container for containing a biological sample of a patient.Also, the kit can also comprise the instructions for using the kit andexplaining the results of the kit.

In the present application, it is found through the experiments thatthere is great difference of the methylation level of RNF180 genebetween gastritis and gastric cancer, and the content of RNF180increases gradually from normal group, chronic superficial gastritisgroup, chronic atrophic gastritis (including chronic gastritiscomplicated with intestinal metaplasia) group, to gastric cancer groupsstages I to IV, while the Ct value decreases gradually. The positiverate also increases gradually from normal group to gastric cancer groupsin the same order. Therefore, the present application provides a methodfor grading gastric cancer and gastritis by measuring the methylationlevel of RNF180 gene in a sample to be tested, thereby providing anoninvasive and rapid method for screening gastric cancer and gastritis.

Septin9 and RNF180 are combined for detecting gastric cancer andassociated with gastric cancer staging (I-IV). RNF180 could also be usedto detect gastritis, and distinguish among normal group, gastritis groupand gastric cancer group based on the significant statistical differenceamong the three groups. In addition, RNF180 could also be used todistinguish detectably the gastritis with intestinal metaplasia fromcommon gastritis without intestinal metaplasia, and the positive ratesof RNF180 of the gastritis with intestinal metaplasia is 100% and commongastritis is about 27%.

The present applicant also finds that the level of methylated RNF180gene in peripheral blood tends to increase as age increases. However,since the elderly has a certain degree of gastritis more or less, it isdifficult to distinguish whether age factor or gastritis factor leads tothe elevated level of methylated RNF180 gene in peripheral blood, whichhas a significant impact on the specificity and reliability of detectionof gastritis and gastric cancer. Therefore, taken this intoconsideration, Septin9 is introduced simultaneously into the detection.As the impact of age factor and gastritis factor on Septin9 gene can beignored, Septin9 can be used to confirm the detection of gastric cancer.Specifically, about 40% of gastric cancer is simultaneously positive forboth Septin9 and RNF180, and the double positives can be used as thecriteria with the specificity of up to 90%. The methylation detection ofSeptin9 can overcome and compensate for the disadvantage of poorspecificity when the methylation detection of RNF180 is used alone, soas to indicate the presence of abnormal cell proliferation moreaccurately. Also, according to certain specific embodiments, for somegastric cancers, RNF180 is negative, but Septin9 is positive, and thesegastric cancers can be detected by Septin9 with the sensitivityincreased by about 3%. The methylation detection of Septin9 can overcomeand compensate for the disadvantage of poor sensitivity when methylationdetection of RNF180 is used alone, so as to indicate the presence ofabnormal cell proliferation more accurately. Thus, both the sensitivityand specificity for grading gastritis and gastric cancer can be improvedby combining the two biomarkers, Septin9 and RNF180.

Finally, the simultaneous bi-channel detection of the two biomarkers,Septin9 and RNF180, can be conveniently achieved by utilizing areal-time PCR assay of DNA in a plasma sample, and whether a sample ispositive or not can be quickly and easily determined according to thecycle threshold (Ct) value of a real-time PCR. The present inventionprovides a non-invasive and rapid method for grading cancer andinflammation.

Unless otherwise defined, the relevant technical and scientific terms inthis specification are intended to be the same meaning as commonlyunderstood by those skilled in the art. Although similar or identicalmethods and materials to those described herein can be applied in theexperimental or practical applications, materials and methods aredescribed herein below. In the case of conflict, reference is made tothe specification including the definitions contained therein. Inaddition, the materials, methods and examples are illustrative only andnot restrictive.

Other features and advantages of the present invention will be describedin detail by the following detailed description and the claims.

BRIEF DESCRIPTION OF FIGURES

The above and other features of the present invention will be furtherdescribed in conjunction with the following figures and their detaileddescription. It should be understood that these figures only showseveral exemplary embodiments in accordance with the present inventionand should not be considered as the limitation of the scope of thepresent invention. Unless otherwise specified, the figures are notnecessarily to be in proportion, and similar reference numbers refer tosimilar parts.

FIG. 1 shows that none of the four sets of primers and probes for RNF180affects the determination of Septin9 in multiple detection of themethylated DNAs of Septin9 and RNF180.

FIG. 2 shows that the positive results of multiple detection of gastriccancer based on the methylated DNAs of Septin9 and RNF180 arecomplementary.

FIG. 3 shows a histogram of the average Ct values of multiple detectionof gastric cancer, gastritis, and normal people based on the methylatedDNAs of Septin9 and RNF180.

FIG. 4 shows a scatter diagram of the Ct values of multiple detection ofgastric cancer, gastritis, and normal people based on the methylatedDNAs of Septin9 and RNF180.

FIG. 5 shows the constant Ct values of β-actin in normal people,superficial, atrophic/intestinal metaplasia, gastric cancer stages I,II, III and IV.

FIG. 6 shows the dCt values of RNF180 in normal people, superficial,atrophic/intestinal metaplasia, and gastric cancer stages I, II, III andIV.

FIGS. 7A-7C show the comparation of RS19 positive rates between normalpeople, superficial, atrophic/intestinal metaplasia, and gastric cancer;the comparation of RS19 positive rates between gastric cancer stages I,II, III and IV; and a histogram of the average dCt values of RS19 ofnormal people, gastritis, and gastric cancer, respectively.

FIG. 8A shows a graph of the relation between the methylation level ofRNF180 and age; FIG. 8B is a schematic diagram showing that thecombination of Septin9 and RNF180 improves the specificity andsensitivity of detection for gastric cancer.

FIG. 9 shows the sensitivity and specificity of multiple detection(RS19) of normal and gastric cancer based on the methylated DNAs ofSeptin9 and RNF180.

FIG. 10 shows the sensitivity and specificity of multiple detection(RS19) of normal and gastritis (chronic, superficial and ulcerative)based on the methylated DNAs of Septin9 and RNF180.

FIG. 11 shows the sensitivity and specificity of multiple detection(RS19) of normal and gastritis (all) based on the methylated DNAs ofSeptin9 and RNF180.

BEST MODES FOR CARRYING OUT INVENTION

Unless otherwise indicated, the implementation of the presentapplication will employ the conventional techniques of molecular biology(including recombinant techniques), microbiology, cell biology,biochemistry, and genomics, all of which fall within the scopes of theconventional technical means in the art. Such techniques are describedin detail in the literatures, such as Molecular Cloning: A LaboratoryManual, 2th Edition (Sambrook et al., 1989); Oligonucleotide Synthesis(M. J. Gait, 1984); Animal Cell Culture (R. I. Freshney, 1987); Methodsin Enzymology series (American Academic Press, Inc.); Current Protocolsin Molecular Biology (F. M. Ausubel et al., 1987, and updatedperiodically); PCR: The Polymerase Chain Reaction (Mullis et al., 1994).The primers, probes and kits used in the present application may beprepared according the standard techniques well known in the art.

Unless otherwise defined, the technical and scientific terms used in thepresent application have the same meaning as commonly understood by oneof the ordinary skilled in the art to which the present inventionbelongs. Singleton et al., Dictionary of Microbiology and MolecularBiology, 2th Edition, J. Wiley & Sons (New York, N.Y. 1994), and March,Advanced Organic Chemistry Reactions, Mechanisms and Structure, 4thEdition, John Wiley & Sons (New York, N.Y. 1992), provide generalguidance to those skilled in the art for a number of terms used in thepresent application.

Definitions

In the present application, “grading disease severity” means that thedisease severity is determined based on detection results, and thusgraded. According to the present application, the two levels, normal andcancer, can be distinguished according to the DNA methylation level,namely, a cancer in an individual can be detected. Preferably, accordingto the present application, the three levels, normal, inflammation andcancer, can be distinguished according to the DNA methylation level. Forexample, the three levels, normal, gastritis and gastric cancer, can bedistinguished according to the methylation level of the DNAs to bemeasured in a sample from a patient, and for gastritis, it can befurther divided into superficial gastritis and atrophic gastritis.

In the present application, “cancer” means and includes any malignancy,or malignant cell division or malignant tumour, or any conditioncomprising uncontrolled or inappropriate cell proliferation, andincludes without limitation any disease characterized by uncontrolled orinappropriate cell proliferation.

In the present application, “gastric cancer” and “stomach cancer” havethe same meaning and mean a cancer of stomach or of stomach cells. Suchcancers may be adenocarcinomas that occur in the lining of stomach(mucosa) and may be in the pylorus, body or cardial (lower, body andupper) parts of stomach.

In the present application, “staging of gastric cancer” means thatgastric cancer can be divided into four stages according to the clinicalpathological staging standard of gastric cancer. The staging of gastriccancer involves three indexes: primary tumor (T), lymph node metastasis(N) and distant metastasis (M, including left supraclavicular lymph nodemetastasis, hematogenous metastasis and intraperitoneal implantation).“Stage I” refers to superficial gastric cancer without lymph nodemetastasis, or although the tumor has invaded muscle layer but notexceeded a half of a subregion; “stage II” refers to superficial cancerwith a first station of lymph node metastasis, T2 cancer (tumor hasinvaded muscle layer of gastric wall but not exceeded a half of asubregion in size) and T3 cancer (tumor has invaded gastric wall serosa,or although the tumor has not invaded the serosa, but the size of thelesion has exceeded a half of a subregion but not more than asubregion), T3 cancer without lymph node metastasis also belongs to thisstage; “stage III” refers to the tumor of various size with a secondstation of lymph node metastasis, or the tumor with only a first stationof lymph node metastasis or of which, the tumor size is exceeded asubregion without lymph node metastasis; and “stage IV” refers to alltumors with a third station of lymph node metastasis or distantmetastasis, regardless of tumor size.

In the present application, “gastric cancer cells” means the cellshaving characteristic of gastric cancer, and includes precancerouscells.

In the present application, “precancerous” means the cells which are inthe early stage of conversion to cancer cells or which is prediposed forconversion to cancer cells. Such cells may show one or more phenotypictraits having characteristic of the cancerous cells.

In the present application, “gastritis” refers to the inflammation ofgastric mucosa caused by any cause, often accompanied with epithelialdamage and cell regeneration. Gastritis is one of the most commondiseases in digestive system, and generally divided into acute gastritisand chronic gastritis according to the urgency degree of its clinicalonset. Chronic gastritis is generally divided into the following types:the gastritis of which inflammatory lesions are relatively superficialand confined to the surface layer of gastric mucosa (not more than athird), known as “superficial gastritis”; and “ulcerative gastritis”, ofwhich the leisions have been developed into gastric ulcers; and“atrophic gastritis”, of which inflammatory lesions spread to the wholegastric mucosa with atrophy of gastric gland. Among these, it is foundin the pathological biopsy of the patients with atrophic gastritis thattwo lesions, “colonic incomplete intestinal metaplasia” and “atypicalhyperplasia” in the gastric mucosa, may develop into gastric cancer.So-called intestinal metaplasia means that small intestinal or colonicgland appears in gastric mucosa glands, that is, intestinal glandmigrates into stomach, which should not occur in normal conditions. Themore the amount of intestinal metaplasia is, the higher the severity ofthe atrophy is. Superficial gastritis and ulcerative gastritis can becollectively referred as “mild gastritis”; and “atrophic gastritis” and“gastritis with intestinal metaplasia” can be collectively referred assevere gastritis.

In the present application, “biomarker” refers to a substance such as agene as a variable related to a disease to be measured, which may serveas an indicator or predictor of the disease. A biomarker may be theparameter from which the presence or risk of a disease can be inferred,without the detection of the disease itself.

In the present application, “nucleic acid”, “nucleic acid sequence” andthe like refers to polynucleotides, which may be gDNA, cDNA or RNA, andsingle-stranded or double-stranded. The term also includes peptidenucleic acid (PNA), or any chemically DNA-like or RNA-like material.“cDNA” refers to a DNA which is copied from naturally occurring mRNA.“gDNA” refers to a genomic DNA. A combination of these materials canalso be included (i.e., a recombinant nucleic acid in which a part isgDNA and another part is cDNA).

In the present application, “operably associated” and “operably linked”refers to functional bond to a nucleic acid sequence.

In the present application, “stringent hybridization conditions” and“high stringency” refer to the conditions under which a probe hybridizesto its target subsequence, typically in a complex mixture of nucleicacids. Stringent conditions are sequence-dependent and different indifferent circumstances. A longer sequence hybridizes specifically at ahigher temperature. An extensive guidance on the hybridization ofnucleic acids is found in Tijssen, Techniques in Biochemistry andMolecular Biology—Hybridization with Nucleic Probes, “Overview ofprinciple of hybridization and strategy of nucleic acid experiment”.Generally, stringent conditions are lower than the melting point (Tm)for a specific nucleic acid by about 5-10° C. at a defined ionicstrength and pH. At a temperature of Tm (with defined ionic strength,pH, and concentration of nucleic acid), 50% of probes complementary to atarget hybridize to a target sequence at equilibrium. Stringentconditions may also be achieved with the addition of a destabilizingagent. For a selective or specific hybridization, a positive signal isat least two times, preferably 10 times of background hybridization.Exemplary stringent hybridization conditions are as following:hybridizing at 42° C. in a solution of 50% formamide, 5×SSC, and 1% SDS,or hybridizing at 65° C. in a solution of 5×SSC, 1% SDS, then washing ina solution of 0.2×SSC and 0.1% SDS at 65° C.

Furthermore, nucleic acids that can not hybridize under stringentconditions are still substantially similar if the polypeptides whichthey encode are substantially similar. In this case, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” includehybridizing in a solution of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and washing in a solution of 1×SSC at 45° C. Those of ordinary skill inthe art can be readily taught from the prior art the conditions underwhich how to achieve identical stringency. For PCR, a temperature ofabout 36° C. is typical for amplification with low stringency, and anannealing temperature may range from 32° C. to 48° C. depending onprimer length. For highly stringent PCR amplification, a temperature ofabout 62° C. is typical, and an annealing temperature of highlystringent hybridization can range from 50° C. to 65° C., depending onprimer length and specificity. Typical cycle conditions for bothamplifications with high and low stringency include a consecutivedenaturation phase at 90° C.−95° C. for 30 sec-2 min., an consecutiveannealing phase for 30 sec-2 min., and an consecutive extension phase atabout 72° C. for 1-2 min. The tools and guidelines for amplificationreactions with low and high stringency can obtained from the prior art.

In the present application, “oligonucleotide” refers to a moleculeconsisting of two or more nucleotides, preferably more than threenucleotides. Its exact size will depend upon many factors which, inturn, depend upon the ultimate function and use of oligonucleotide. Incertain specific embodiments, an oligonucleotide may have a length of 10to 100 nucleotides. In certain specific embodiments, an oligonucleotidemay have a length of about 10 to 30 nucleotides, or may have a length ofabout 20 to 25 nucleotides. In some particular embodiments, anoligonucleotide having a shorter length may be suitable.

In the present application, “primer” means an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a initiation point ofsynthesis when placed under conditions in which synthesis of a primerextension product, which is complementary to a nucleic acid strand, isinduced, i.e., in the presence of nucleotides and an inducing agent suchas a DNA or RNA polymerase and at a suitable temperature and pH. Theprimer may be either single-stranded or double-stranded and must besufficiently long to prime the synthesis of the desired extensionproduct in the presence of an inducing agent. The exact length of aprimer will depend upon many factors, including temperature, primersource and method to be used. For example, for diagnostic and prognosticapplications, depending on the complexity of a target sequence,oligonucleotide primers typically contain at least or more than about10, or 15, or 20, or 25 or more nucleotides, although it may containfewer or more nucleotides. The factors involved in determining theappropriate length of a primer are well known to a person skilled in theart.

In the present application, “primer pair” means a pair of primers whichhybridize to the opposite strands of a target DNA molecule, or to theregions of a target DNA which flank a nucleotide sequence to beamplified.

In the present application, “primer site” means the region of a targetDNA or other nucleic acid to which a primer hybridizes.

In the present application, “probe” with regard to a nucleic acidsequence is used in its ordinary sense to mean a selected nucleic acidsequence that can hybridise under specified conditions to a targetsequence and may be used to detect the presence of such target sequence.It will be understood by those skilled in the art that in some instancesprobes may be also be used as primers, and primers may be used asprobes.

In the present application, “DNA methylation” refers to the addition ofa methyl group to the 5-position of cytosine (C), typically (but notnecessarily) in the context of CpG (cytosine followed by guanine)dinucleotides. As used herein, “an increased methylation level” or “asignificant methylation level” refers to the presence of at least onemethylated C nucleotide in a DNA sequence where the corresponding C isnot methylated in a normal control sample (such as a DNA sampleextracted from a non-cancerous cell or tissue sample, or a DNA samplethat has been subjected to a treatment of methylation on DNA residues),in some embodiments at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more Cs maybe methylated at locations where the Cs are unmethylated in a controlDNA sample.

In the embodiments, an alteration of DNA methylation can be detected bya number of different methods. The methods for detecting DNA methylationinclude, for example, an assay of methylation-sensitive restrictionendonuclease (MSREs) by either southern or polymerase chain reaction(PCR) analysis, methylation specific or sensitive PCR (MS-PCR),methylation-sensitive single nucleotide primer extension (Ms-SnuPE), ahigh resolution melting (HRM) analysis, bisulifte sequencing,pyrosequencing, methylation-specific single-strand conformation analysis(MS-SSCA), combined bisulifte restriction analysis (COBRA),methylation-specific denaturing gradient gel electrophoresis (MS-DGGE),methylation-specific melting curve analysis (MS-MCA),methylation-specific denaturing high-performance liquid chromatography(MS-DHPLC), methylation-specific microarray (MSO). These assays can beeither PCR analysis, quantitative analysis with fluorescence labellingor southern blot analysis.

In the present application, “determination of methylation” means anydetermination that confirms the methylation status of one or more CpGdinucleotide sequence(s) within a DNA sequence.

In the present application, “biological sample” or “sample” includessections of tissues, such as biopsy and autopsy samples, and frozensections taken for histologic purposes, or processed forms of any ofsuch samples. Biological samples include blood and blood fractions orproducts (e.g., serum, plasma, platelets, red blood cells, and thelike), sputum or saliva, lymph and tongue tissues, cultured cells, e.g.primary cultures, explants, and transformed cells, stool, urine, stomachbiopsy tissues, etc. A biological sample is typically obtained from aeukaryotic organism, which may be a mammal, may be a primate and may bea human individual.

In the present application, “biopsy” refers to the process of removing atissue sample for diagnostic or prognostic evaluation, and to the tissuespecimen itself. Any biopsy technique known in the art can be applied tothe diagnostic and prognostic methods of the present invention. Thebiopsy technique applied will depend on the tissue type to be evaluated(e.g., tongue, colon, prostate, kidney, bladder, lymph node, liver, bonemarrow, blood cell, stomach tissue, etc.) among other factors.Representative biopsy techniques include, but are not limited to,excisional biopsy, incisional biopsy, needle biopsy, surgical biopsy,and bone marrow biopsy. Colonoscopy is also included. A wide range ofbiopsy techniques are well known to those skilled in the art who willchoose from them and implement them with minimal experimentation.

In the present application, “isolated” nucleic acid molecule means anucleic acid molecule that is separated from other nucleic acidmolecules that are usually associated with the isolated nucleic acidmolecule. Thus, an “isolated” nucleic acid molecule includes, withoutlimitation, a nucleic acid molecule that is free of sequences thatnaturally flank one or both ends of the nucleic acid in the genome ofthe organism from which the isolated nucleic acid is derived (e.g., acDNA or genomic DNA fragment produced by PCR or restriction endonucleasedigestion). Such an isolated nucleic acid molecule is generallyintroduced into a vector (e.g., a cloning vector, or an expressionvector) for convenience of manipulation or to generate a fusion nucleicacid molecule. In addition, an isolated nucleic acid molecule caninclude an engineered nucleic acid molecule such as a recombinant orsynthetic nucleic acid molecule. A nucleic acid molecule existing amonghundreds to millions of other nucleic acid molecules within, forexample, a nucleic acid library (e.g., a cDNA or genomic library) or aportion of a gel (e.g., agarose, or polyacrylamide) containingrestriction-digested genomic DNA is not to be considered an isolatednucleic acid.

In the present application, “cell” may be isolated, may be comprised ina population of cells, may be in culture, or may be comprised in aliving individual and may be a mammalian cell and may be a human cell.Similarly, “tissue” may comprise any number of cells and may becomprised in a living individual or may be isolated therefrom.

In the present application, “purified” or “substantially purified” withregard to nucleic acids or polypeptides means those separated from theirnatural environment so that they consitute at least about 75%, 80%, 85%,90% or 95% of total nucleic acid or polypeptide or organic chemical in agiven sample. Protein purity is assessed herein by SDS-PAGE and silverstaining. Nucleic acid purity is assessed by agarose gel and EtBrstaining.

In the present application, “detection” means any process of observing amarker, or a change in a marker (such as for example the change in themethylation status of a marker, or the expression level of a nucleicacid or protein sequences), in a biological sample, whether or not themarker or the change in the marker is actually detected. In other words,the act of probing a sample for a marker or a change in a marker is a“detection”, even if the marker is determined to be not present or belowthe level of sensitivity. Detection may be a quantitative,semi-quantitative or non-quantitative observation and may be based on acomparison with one or more control samples. It will be understood thatdetecting a gastric cancer as disclosed herein includes detectingprecancerous cells that are beginning to or will, or have an increasedpredisposition to develop into gastric cancer cells. Detecting a gastriccancer also includes determining possible probability of mortality or alikely prognosis for the condition.

In the present application, “homology”, “identity” and “similarity” meansequence similarity between two nucleic acid molecules. They can each bedetermined by comparing a position in each sequence which may be alignedfor purposes of comparison. When an equivalent position in the comparedsequences is occupied by the same base, then the molecules are identicalat that position; when the equivalent site occupied by the same or asimilar amino acid residue (e.g., similar in steric and/or electronicnature), then the molecules can be referred to as homologous (similar)at that position. Expression as a percentage of homology/similarity oridentity refers to a function of the number of identical or similaramino acids at positions shared by the compared sequences. A sequencewhich is “unrelated” or “non-homologous” shares less than 40% identity,preferably less than 25% identity with a sequence of the presentinvention. In comparing two sequences, the absence of residues (aminoacids or nucleic acids) or presence of extra residues also decreases theidentity and homology/similarity. In specific embodiments, two or moresequences or subsequences may be considered substantially orsignificantly homologous, similar or identical if the identity betweensequences are about 60%, or are about 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more over a specified region,when compared and aligned for maximum correspondence over a comparisonwindow or designated region, as measured using a BLAST or BLAST 2.0sequence comparison algorithms with default parameters described below,or by manual alignment and visual inspection such as provided on-line bythe National Center for Biotechnology Information (NCBI). Thisdefinition also refers to, or may be applied to, the compliment of atest sequence. Thus, to the extent allowed in the context herein, forexample, if a nucleotide sequence can be predicted to be naturallyoccurring in a DNA duplex, or naturally occurring as one or two ofcomplementary strands, the nucleotide sequence itself which iscomplementary to a specified target sequence or a variant thereof isconsidered to be “similar” to the target sequence, and when referring toa “similar” nucleic acid sequence, it includes a single strandedsequence, its complementary sequence, a double stranded chain complex, asequence capable of encoding an identical or similar polypeptideproduct, and any permissible variants of any described above. The casewhere the similarity must be limited to the analysis of a single nucleicacid strand sequence may include, for example, the detection andquantification of the expression of a particular RNA sequence orencoding sequence in a cell. The definition also includes a sequencethat has deletion and/or addition, as well as has a substitution. In theembodiments, identity or similarity exists over a region of at leastabout 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore nucleotides in length, or over a region of more than about 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or morethan 100 nucleotides in length.

In the present application, “amplification” means a process wherebymultiple copies are obtained from one specific locus of a nucleic acid,such as a genomic DNA or cDNA. Amplification can be accomplished by anyone of a number of known means, including but not limited to apolymerase chain reaction (PCR), transcription-based amplification andstrand displacement amplification (SDA).

In the present application, “standard amplification conditions” refersto the basic components of an amplification reaction mixture and cyclingconditions that include multiple cycles of denaturing template nucleicacid, annealing the oligonucleotide primers with template nucleic acid,and extending primers by a polymerase to produce an amplificationproduct.

In the present application, “polymerase chain reaction” or “PCR” means atechnique in which cycles of denaturation, annealing with primers, andextension with a DNA polymerase are used to amplify the number of copiesof a target DNA sequence by approximately 10⁶ times or more. Apolymerase chain reaction process for amplifying nucleic acid is foundin U.S. Pat. Nos. 4,683,195 and 4,683,202.

In the present application, “fluorescence-based real-time PCR” means amethod in which a fluorophore is added to a PCR reaction system tomonitor the progress of a whole PCR process in real time by fluorescencesignal accumulation, and an unknown template is quantitatively analyzedby a standard curve. In the PCR technique, there is a very importantconcept, i.e., cycle threshold, also known as Ct value. C representscycle, and t represents threshold (critical value). Ct value means thatthe number of cycles experienced when the fluorescence signal in eachreaction tube reaches the set threshold value. For example, afluorescence threshold is set as follow: the fluorescence signal of thefirst 15 cycles of a PCR reaction is used as the fluorescence backgroundsignal and the default setting of the fluorescence threshold is 10 timesof the standard deviation of the fluorescence signal from 3 to 15cycles.

In the present application, “cut-off value of real-time PCR” representsone critical Ct value for determining that a given sample is positive ornegative for one certain biomarker. According to certain specificembodiments of the present application, the “critical Ct value (cut offvalue)” is obtained according to a certain number of sample data andbased on a statistical treatment, which may vary depending on thedesired sensitivity or specificity requirements. In the summary, thiscritical Ct value will be further illustrated.

In the present application, “sensitivity” means the ratio of cancersdetected from certain cancer samples and is calculated as:Sensitivity=(detected cancers/all cancers), and “specificity” means theratio of normal detected from certain normal samples and is calculatedas: Specificity=(undetected negatives/total negatives).

In the present application, “label” or “detectable moiety” is acomponent detectable by spectroscopic, photochemical, biochemical,immunochemical, chemical, or other physical means. For example, usefullabels include 32P, fluorescent dyes, electron-dense reagents, enzymes(e.g., those commonly used in an ELISA), biotin, digoxigenin, or haptensand proteins which can be made detectable, e.g., by incorporating aradiolabel into a peptide or antibodies used to detect specificalreaction with a peptide.

Nucleic acid molecules can be detected using a number of differentmethods. Methods for detecting nucleic acids include, for example, PCRand nucleic acid hybridizations (e.g., Southern blot, Northern blot, orin situ hybridizations). Specifically, oligonucleotides (e.g.,oligonucleotide primers) capable of amplifying a target nucleic acid canbe used in a PCR reaction. PCR methods generally include the steps ofobtaining a sample, isolating nucleic acid (e.g., DNA, RNA, or both)from the sample, and contacting the nucleic acid with one or moreoligonucleotide primers that hybridize(s) with specificity to thetemplate nucleic acid under conditions under which amplification of thetemplate nucleic acid can occur. In the presence of a template nucleicacid, an amplification product is produced. Conditions for amplificationof a nucleic acid and detection of an amplification product are known tothose of skill in the art. A range of modifications to the basictechnique of PCR also have been developed, including but not limited toanchor PCR, RACE PCR, RT-PCR, and ligation chain reaction (LCR). A pairof primers in an amplification reaction must anneal to opposite strandsof the template nucleic acid, and should be an appropriate distance fromone another such that the polymerase can effectively polymerize acrossthe region and such that the amplification product can be readilydetected using, for example, electrophoresis. Oligonucleotide primerscan be designed using, for example, a computer program such as OLIGO(Molecular Biology Insights Inc., Cascade, Colo.) to assist in designingoligonucleotide primers that have similar melting temperatures.Typically, oligonucleotide primers are 10 to 30 or 40 or 50 nucleotidesin length (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length), but can belonger or shorter if appropriate amplification conditions are used.

Detection of an amplification product or a hybridization complex isusually accomplished using detectable labels. The term “label” withregard to a nucleic acid is intended to encompass direct labeling of anucleic acid by coupling (i.e., physically linking) a detectablesubstance to the nucleic acid, as well as indirect labeling of thenucleic acid by reactivity with another reagent that is directly labeledwith a detectable substance. Detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin. Anexample of indirect labeling includes end-labeling a nucleic acid withbiotin such that it can be detected with fluorescently labeledstreptavidin.

SUMMARY

The present application provides a detection method for a cancer bycombining Septin9 and RNF180 genes, and the corresponding composition,kit and nucleic acid sequences, for non-invasive diagnosis and detectionof a cancer with high efficiency and sensitivity. It has been foundaccording to the specific embodiments that the combination of Septin9and RNF180 greatly improves the sensitivity or specificity of detectionof a cancer, particularly of gastric cancer.

The present application also provides a method for grading diseaseseverity by RNF180 gene, preferably a combination of Septin9 and RNF180genes, as well as the corresponding composition, kit and nucleic acidsequences, so that a cancer and inflammation, especially gastric cancerand gastritis be can be graded efficiently and sensitively in anoninvasive manner.

The followings are the examples of the composition, kit, nucleic acidsequences, and detection methods of the present application. It will beunderstood that a variety of other embodiments could be implemented inview of the general description provided above.

In the first group of embodiments, a composition for diagnosing ordetecting abnormal cell proliferation in a biological sample isdisclosed, which includes a nucleic acid for detecting the methylationlevel within at least one target region of Septin9 and RNF180 genes orfragments thereof. In particular, the composition comprises not only anucleic acid sequence for detecting the methylation level within atleast one target region of Septin9 gene or fragment thereof, but also anucleic acid sequence for detecting the methylation level within atleast one target region of RNF180 gene or fragment thereof.

The present application also discloses a composition for grading diseaseseverity in an individual, which comprises a nucleic acid for detectingthe methylation level within at least one target region of RNF180 geneor fragment thereof, so as to grade disease severity based on thedetection result of the methylation of RNF180. Preferably, thecomposition comprises not only a nucleic acid sequence for detecting thepresence or absence of methylation within at least one target region ofRNF180 gene or fragment thereof, but also a nucleic acid sequence fordetecting the presence or absence of methylation within at least onetarget region of Septin9 gene or fragment thereof.

In the following, Septin9 gene will be introduced firstly. Human Septin9gene (also known as MLL septin-like fusion protein, MLL septin-likefusion protein MSF-A, Slpa, Eseptin, Msf, septin-like ovarian/breastseptin (Ov/Brseptin), and Septin D1), which is a member of the Septingene family, located within contig AC068594.15.1.168501 of chromosome17q25. For example, SEQ ID No: 13 provides a sequence of promoter regionof Septin9 which is rich in CpG.

The sequence of SEQ ID No: 13 is as follow:

CGTTACCCGAGTTGTAAAGGGCGGCTCCCTGTGTCTGCCCCGCTGCACCGATACACCGAGCTGCGCACGGTGCCCAGCGCAGGGAGAACAAATGATCATCTGTCCAACGCGCCCATTTACAGGTGAGGAAACTAAGGCTCCAACTCAATCGACGCACTCTGCCCTTTTGATTACCAGAAAAGTAGCAGGACAGGTGTCCTGTCCCGCCCTACCCCGGCCCACTAAGCCGGCACCCCGGCTCCGACCCCCGGCTGTGCCCGGCGCCGCCGCGGTGCCCGGCGCCGCCGCCTCGCCCGGCGGGGCCGCCCGGAGCGCCCGCACCTCCGCCCGCTTCCACCTGGCCGGGCCCGCCCCGCCCGGACTCGGGACTGGGAAGTGCGGCGACTCCCGGAACCAGCCATTGGCGCCAGCGCGGGGAGCTGGGGGTGCAGAGCTGCGGGCGCGGCGGGCCACGCAGGCGGCCCCCACCCCCGGCCTGGCCTGGTCTGGTCTGGTCTGCGCTGCCGCGCGGGGGCGCCCCCTCCCAGGCCCGGCGCCCGCCAGCCCCGCTCCGCCAGGTGCAGCGCAGCGCAGGGGTGGGCGGGGGTGGGGCTCGGCGCGCACGTTCACGGGGCGGGGAGGGGGCGGGTCAGGGGCGGGACCACAGCCGGCTGGGCCGGGGTTCTATGCGCATCTCCGGGGAGGGGCGGGGCGGGGGCGGGGCCGGGGCGGGGCCCGGTCGGTGCACTCCAGACGGCGGGCCGCCCCCTCTTCCCGCCTTCCTACTACCGGCCCAGGATTAGCGCCCTGGGAGCGCGCGCCCCGCTGCCTCGCCGCCACACTTTCCTGGGAGCGGCGGCCACGGAGGCACCATGAAGAAGTCTTACTCAGGTGGGCTTCGCGCCCGGGGTGGGGAGGGGTCGGTGTCCCGGGACCAGCGCTGCTCACCTGAGTGCCTGCGGCCGGGAGTGGCGAGGCGCCCCCGGAGCTGAGCGAGTCCCCGCGGCGGGCACACTGCAGGTCGAGTTCCTCCCAGGACAGGGCCGCTGTCGGGCCGCTTTCGACCTGAGCCGACCGTCCCCTGCGCTGTCTCCAGCCCTTGCTCGAGTGTCGGAGGGGCTGCCCTGGGGGACGCTCCCTCTTCCTCGCCCCTTGCACCCTCGCAGGAATCGCTGACTTTCCAGGTCGGCCGGGTGCTTTGGGTCCCTGTGCGTCTGTGTGGGTGAATGGGGTCGGGGCTAGGTGGAGGGGTGTCCTTGGGTTCAGCCTCTAGGGCTGGTGGTCCAGGCCGCAGCATCCTTTCTTCGGATTCTCTTCGGTTTCTCCTCTACTTAGTGGGGCACGGGACGGCCTCCAGATGGGACCGTCCAGCAGCGCCCAAACTTGGCGACTCGGGTTCACGTTTTGCGCTCAGGACGCCG CCCGC

The members of the Septin gene family are involved in multiple cellularfunctions ranging from vesicular transport to cytokinesis. Thedestructive effect on Septin 9 results in incomplete cell division, andSeptin 9 and other proteins have been shown to be fusion partners ofprotooncogene MLL, indicating their roles in tumorigenesis.

The nucleic acid sequence for detecting the presence or absence ofmethylation within at least one target region of Septin9 gene orfragment thereof comprises a long fragment of at least 9 bases which isequivalent to, complementary to, or hybridizes under moderatelystringent or stringent conditions to a contiguous sequence selected fromSEQ ID No: 13.

According to a specific embodiment, the nucleic acid sequence fordetecting the presence or absence of methylation within at least onetarget region of RNF180 gene or fragment thereof may comprises at least15 nucleotides which are equivalent to, complementary to, or hybridizeunder moderately stringent or stringent conditions to SEQ ID No: 10 asshown below, or complementary sequence thereof, and SEQ ID No: 10represents the promoter region of RNF180 and corresponds to Genbankaccession number: NMi001113561 chr51 63497153-63497758. The underlinedindicates the “transcription initiation site”.

According to certain preferred embodiments, the primers and probes canbe designed based on the sequence of SEQ ID No: 10. The sequencessuitable for serving as the primers and probes for PCR amplification maycomprise nucleotides of any suitable length, e.g., may comprise at least15 nucleotides, or may comprise at least 20, 25, 30, 35, 40, 45, or morethan 50 nucleotides. In these specific embodiments, the nucleic acidsequence may have similarity of approximately 95%, 96%, 97%, 98%, or 99%to the sequence of SEQ ID No: 10 or complementary sequence thereof.

SEQ ID No: 10 (−234 bp to +372 bp with respect to the transcriptioninitiation site)

GATAATTTCTGTGGCTCTGGTAAGGGGATGACAAGGGAGAAAAACTTTCCCACGGTTCCGTCTGGCCCGCGGCGCTTGTCTGCCTGCGCGGGGTCAAAGCCCGGCGCCGCCCACGCGCGGCTCGGGTGGGAACCCGCAGACGTGGGGCGAGCAGGGCCGCTGGCTGTGGCGGGCGAGCGCCGGGGCGCCACGTCCGAGGCCGCGGGGTCGGGGCTGCAGGCACAGCTCGAGCGCTTTCCGCGGGGTTTGGCTCCTGTCGCTTCCCGTCTCGCCGAACCGGCATCGCCGCCGCCGGAGCCGCAGCGAGTCCTCAGAGCCTGGCTGCTGGCGGCCGGGAGCGCCGGGACGGGGGCGCGAAGCCGGAGGCTCCGGGACGTGGATACAGGTAAAGGCCGGCGGGTCGGAGTCGGGCGGGGCGCGGCGGCGGCGCCTCTCGGAGGGACCTGGCCTCGGCCGGGCCCTACCCAGCCGCGGTGGCCCGGGCCCCCACGTTGGCCCAGGCGGGGACGTGCCAAGGGGCTGGGCTAGGGTTGCCGCTGGCCTGGCCGCCTCTCGCCCGGCGGGCCTCAGGTGACGCGGCCGCGGCTTAACTTTCGCACC TGAGGCT

Preferably, the nucleic acid sequence used to detect the presence orabsence of methylation within at least one target region of RNF180 geneor fragment thereof comprises at least 15 nucleotides which areequivalent to, complementary to, or hybridize under moderately stringentor stringent conditions to SEQ ID No: 11 shown below, or complementarysequence thereof.

SEQ ID No: 11 (−167 bp to +135 bp with respect to the transcriptioninitiation site)

CGCGGCGCTTGTCTGCCTGCGCGGGGTCAAAGCCCGGCGCCGCCCACGCGCGGCTCGGGTGGGAACCCGCAGACGTGGGGCGAGCAGGGCCGCTGGCTGTGGCGGGCGAGCGCCGGGGCGCCACGTCCGAGGCCGCGGGGTCGGGGCTGCAGGCACAGCTCGAGCGCTTTCCGCGGGGTTTGGCTCCTGTCGCTTCCCGTCTCGCCGAACCGGCATCGCCGCCGCCGGAGCCGCAGCGAGTCCTCAGAGCCTGGCTGCTGGCGGCCGGGAGCGCCGGGACGGGGCGCGAAGCCGGAGGCT CC

Further preferably, the nucleic acid sequence used to detect thepresence or absence of methylation within at least one target region ofRNF180 gene or fragment thereof comprises at least 15 nucleotides whichare equivalent to, complementary to, or hybridize under moderatelystringent or stringent conditions to SEQ ID No: 12 shown below, orcomplementary sequence thereof.

SEQ ID No: 12 (−43 bp to +135 bp with respect to the transcriptioninitiation site)

GTCCGAGGCCGCGGGGTCGGGGCTGCAGGCACAGCTCGAGCGCTTTCCGCGGGGTTTGGCTCCTGTCGCTTCCCGTCTCGCCGAACCGGCATCGCCGCCGCCGGAGCCGCAGCGAGTCCTCAGAGCCTGGCTGCTGGCGGCCGGGAGCGCCGGGACGGGGCGCGAAGCCGGAGGCTCC

Therefore, TaqMan probes and primers can be designed to detect the DNAmethylation of the promoter region of RNF180 gene (−234 bp to +372 bpwith respect to the transcription initiation site), preferably thepromoter region to be detected is −167 bp to +135 bp with respect to thetranscription initiation site, more preferably the promoter region to bedetected is −43 bp to +135 bp with respect to the transcriptioninitiation site. The length of an amplicon for detection ranges from 66bp to 130 bp.

For example, in certain specific embodiments, the primers and probes aredesigned to detect the presence or absence of methylation within atleast one target region of RNF180 gene or fragment thereof with SEQ IDNo: 12 (−43 bp to +135 bp with respect to the transcription initiationsite) as the target region. Therefore, a variety of combinations ofprobes and primers can be designed according to the present application,and each combination of probes and primers may be different in terms ofperformance. Therefore, in order to screen the primers and probes withhigh efficiency, the present application utilizes an artificiallymethylated template and an unmethylated template, as well as cancer(e.g., gastric cancer) and normal DNA as templates, to screen themultiple designed combinations of probes and primers by the followingsteps:

1. designing the primers and probes for the promoter region of RNF180gene;

2. designing an artificially methylated DNA and an unmethylated DNA;

3. screening the primers and probes with the artificially methylated DNAand the unmethylated DNA, wherein the artificially methylated DNA isamplified, while the artificially unmethylated DNA is not amplified;

4. screening the primers and probes from the DNA extracted from normalleukocyte DNA.

5. screening the primers and probes with the DNA extracted from humangastric cancer tissue if there is little or no amplification of the DNAextracted from of normal leukocytes.

6. screening the primers and probes from the DNA extracted from normalplasma at clinical study phase;

7. screening the primers and probes with the DNA extracted from thepatients with gastric cancer if there is little or no amplification innormal plasma at clinical study phase.

Through the screening described above, the following sequences of SEQ IDNos: 1-9 are constructed as primers and probes:

Primer: SEQ ID No: 1 (180F7) 5′-GTTCGAGGTCGCGGGGTC-3′ Probe:SEQ ID No: 2 (180P7) 5′- CAL Fluor Red-AACGCTCGAACTATACCTACAACCCC-BHQ2-3′ Primer: SEQ ID No: 3(180R7) 5′-ACAAAAACCAAACCCCGCG-3′ Primer: SEQ ID No: 4(180F24) 5′-GCGGGGTTTGGTTTTTGT-3′ Probe: SEQ ID No: 5(180P2) 5′-CAL Fluor Red-CCGACGACGACGATACCG- BHQ2-3′ Primer:SEQ ID No: 6 (180R2) 5′-ACAACCAAACTCTAAAAACTCG-3′ Probe: SEQ ID No: 7(180P14) 5′-CAL Fluor Red-CGTCGGAGTCGTAGCGAGTTT- BHQ2-3′ Primer:SEQ ID No: 8 (180R135) 5′-AAAACCTCCAACTTCACACCC-3′ Primer: SEQ ID No: 9(180R14) 5′-CGCCAACAACCAAACTCTAA-3′

Further, the present applicant has designed at least one Primers andprobes combination based on the screened primers and probes describedabove, among which the following four combinations are preferable:

-   -   A. Primers and probes combination 1 (amplicon 66 bp, −43 bp to        +23 bp with respect to the transcription initiation site)

SEQ ID No: 1 (180F7) 5′-GTTCGAGGTCGCGGGGTC-3′ SEQ ID No: 2(180P7) 5′- CAL Fluor Red- AACGCTCGAACTATACCTACAACCCC-BHQ2-3′SEQ ID No: 3 (180R7) 5′-ACAAAAACCAAACCCCGCG-3′

-   -   B. Primers and probes combination 2 (amplicon 86 bp, +5 bp to        +91 bp with respect to the transcription initiation site)

SEQ ID No: 4 (180F24) 5′-GCGGGGTTTGGTTTTTGT-3′ SEQ ID No: 5(180P2) 5′- CAL Fluor Red-CCGACGACGACGATACCG- BHQ2-3′ SEQ ID No: 6(180R2) 5′-ACAACCAAACTCTAAAAACTCG-3′

-   -   C. Primers and probes combination 3 (amplicon 130 bp, +5 bp to        +135 bp with respect to the transcription initiation site)

(180F24) SEQ ID No: 4 5′-GCGGGGTTTGGTTTTTGT-3′ (180P14) SEQ ID No: 75′-CAL Fluor Red-CGTCGGAGTCGTAGCGAGTTT-BHQ2-3′ (180R135) SEQ ID No: 85′-AAAACCTCCAACTTCACACCC-3′

-   -   D. Primers and probes combination 4 (amplicon 91 bp, +5 bp to        +96 bp with respect to the transcription initiation site)

(180F24) SEQ ID No: 4 5′-GCGGGGTTTGGTTTTTGT-3′ (180P14) SEQ ID No: 75′-CAL Fluor Red-CGTCGGAGTCGTAGCGAGTTT-BHQ2-3′ (180R14) SEQ ID No: 95′-CGCCAACAACCAAACTCTAA-3′

The sites of the above-described combinations of primers and probesbinding to the above-described gene sequences are further illustratedbelow (wherein the underlined by the lines of the same type indicatesthe corresponding parts).

A. Primers and probes combination 1 (amplicon 66 bp, −43 bp to +23 bpwith respect to the transcription initiation site)

GATAATTTCTGTGGCTCTGGTAAGGGGATGACAAGGGAGAAAAACTTTCCCACGGTTCCGTCTGGCCCGCGGCGCTTGTCTGCCTGCGCGGGGTCAAAGCCCGGCGCCGCCCACGCGCGGCTCGGGTGGGAACCCGCAGACGTGGGGCGThe schematic diagram of the binding sites:

(180F7) SEQ ID No 1 5′-GTTCGAGGTCGCGGGGTC-3′AGCAGGGCCGCTGGCTGTGGCGGGCGAGCGCCGGGGCGCCACGTCCGAGG CCGCGGGGTCGGGGCTGCAGGC (180P7) SEQ ID No 2 5′-CAL Fluor Red- AACGCTCGAACTATACCTACAACCCC-BHQ2-3′ACAGCTCGAGCGCTTTCCGCGGGGTTTGGCTCCTGTCGCTTCCCGTCTCGCCGAACCGGCATCGCCGCCGCCGGAG (180R7) SEQ ID No 3 5′-ACAAAAACCAAACCCCGCG-3′CCGCAGCGAGTCCTCAGAGCCTGGCTGCTGGCGGCCGGGAGCGCCGGGACGGGGCGCGAAGCCGGAGGCTCCGGGACGTGGATACAGGTAAAGGCCGGCGGGTCGGAGTCGGGCGGGGCGCGGCGGCGGCGCCTCTCGGAGGGACCTGGCCTCGGCCGGGCCCTACCCAGCCGCGGTGGCCCGGGCCCCCACGTTGGCCCAGGCGGGGACGTGCCAAGGGGCTGGGCTAGGGTTGCCGCTGGCCTGGCCGCCTCTCGCCCGGCGGGCCTCAGGTGACGCGGCCGCGGCTTAACTTTCGCACCTGAG GCT

B. Primers and probes combination 2 (amplicon 86 bp, +5 bp to +91 bpwith respect to the transcription initiation site)

GATAATTTCTGTGGCTCTGGTAAGGGGATGACAAGGGAGAAAAACTTTCCCACGGTTCCGTCTGGCCCGCGGCGCTTGTCTGCCTGCGCGGGGTCAAAGCCCGGCGCCGCCCACGCGCGGCTCGGGTGGGAACCCGCAGACGTGGGGCGAGCAGGGCCGCTGGCTGTGGCGGGCGAGCGCCGGGGCGCCACGTCCGAGGC CGCGGGGTCGGGGCTGCAGGCThe schematic diagram of the binding sites:

GGGCGCGAAGCCGGAGGCTCCG

C. Primers and probes combination 3 (amplicon 130 bp, +5 bp to +135 bpwith respect to the transcription initiation site)

GATAATTTCTGTGGCTCTGGTAAGGGGATGACAAGGGAGAAAAACTTTCCCACGGTTCCGTCTGGCCCGCGGCGCTTGTCTGCCTGCGCGGGGTCAAAGCCCGGCGCCGCCCACGCGCGGCTCGGGTGGGAACCCGCAGACGTGGGGCGAGCAGGGCCGCTGGCTGTGGCGGGCGAGCGCCGGGGCGCCACGTCCGAGGC CGCGGGGTCGGGGCTGCAGGCThe schematic diagram of the binding sites:

GGACGTGGATACAGGTAAAGGCCGGCGGGTCGGAGTCGGGCGGGGCGCGGCGGCGGCGCCTCTCGGAGGGACCTGGCCTCGGCCGGGCCCTACCCAGCCGCGGTGGCCCGGGCCCCCACGTTGGCCCAGGCGGGGACGTGCCAAGGGGCTGGGCTAGGGTTGCCGCTGGCCTGGCCGCCTCTCGCCCGGCGGGCCTCAGGTGACGCGGCCGCGGCTTAACTTTCGCACCTGAGGCT

D. Primers and probes combination 4 (amplicon 91 bp, +5 bp to +96 bpwith respect to the transcription initiation site)

GATAATTTCTGTGGCTCTGGTAAGGGGATGACAAGGGAGAAAAACTTTCCCACGGTTCCGTCTGGCCCGCGGCGCTTGTCTGCCTGCGCGGGGTCAAAGCCCGGCGCCGCCCACGCGCGGCTCGGGTGGGAACCCGCAGACGTGGGGCGAGCAGGGCCGCTGGCTGTGGCGGGCGAGCGCCGGGGCGCCACGTCCGAGGC CGCGGGGTCGGGGCTGCAGGCThe schematic diagram of the binding sites:

GGACGTGGATACAGGTAAAGGCCGGCGGGTCGGAGTCGGGCGGGGCGCGGCGGCGGCGCCTCTCGGAGGGACCTGGCCTCGGCCGGGCCCTACCCAGCCGCGGTGGCCCGGGCCCCCACGTTGGCCCAGGCGGGGACGTGCCAAGGGGCTGGGCTAGGGTTGCCGCTGGCCTGGCCGCCTCTCGCCCGGCGGGCCTCAGGTGACGCGGCCGCGGCTTAACTTTCGCACCTGAGGCT

In certain specific embodiments, the composition further comprises areagent that converts 5-unmethylated cytosine base of a gene to uracilor other base that is detectably different from cytosine in terms ofhybridization performance. For example, the reagent may be bisulfite.

In certain specific embodiments, the abnormal cell proliferation is acancer. For example, the abnormal cell proliferation is gastric cancer.

In certain specific embodiments, the disease severity is divided intothree levels, wherein the first level is normal, the second level isinflammation and the third level is cancer. Preferably, the inflammationis gastritis, and the cancer is gastric cancer.

Further, for the use described or suggested above in the presentapplication, in the second group of embodiments, disclosed is a kitwhich comprises the composition for diagnosing or detecting a biologicalsample as disclosed in the first group of embodiments. The descriptionof the composition is similar to that of the first group of embodimentsand will not be repeated herein.

Such a kit may include a supporter which is divided for hermeticallyaccommodating one or more containers, such as vials, tubes, etc., andeach container comprises a separate element to be used in the method.For example, one of the containers may comprise probes which are or maybe detectably labeled.

Typically, the kit of the present application will comprise a containerfor containing a biological sample of a patient and the instructions forusing the kit and explaining the results of the kit, and particularly,the kit of the present application comprises the materials as requiredfrom a commercial and user's perspective, i.e, a container forcontaining a biological sample of a patient, a buffer, a diluent, afilter, a needle, a syringe, and instructions inserted in a package. Alabel may be used on the container to indicate that the components areused for a paticular therapeutic or non-therapeutic application, and canalso be used in vivo or in vitro, such as those described above.

There is a variety of embodiments for the kit of the presentapplication. A typical embodiment is a kit which comprises a container,a label on the container, and components within the container; whereinthe components comprise a nucleic acid for detecting the methylationlevel within at least one target region of Septin9 and RNF180 genes orfragments thereof. The label on the container indicates that themethylation level of the DNA of a sample can be evaluated with thecomponents and the instructions on how to use the kit. The kit mayfurther comprise a set of instructions and materials for preparingtissue sample and applying the composition of the present application tothe sample. The kit may comprise a reagent which converts 5-unmethylatedcytosine base of a gene to uracil or other base that is detectablydifferent from cytosine in terms of hybridization performance, such asbisulfite.

In the third group of embodiments, disclosed is a method for detectingabnormal cell proliferation in an individual, comprising determining themethylation level within at least one target region of RNF180 andSeptin9 genes or fragment thereof in a biological sample isolated fromthe individual, and detecting the abnormalcell proliferation in theindividual by combining the detection results of the methylation ofRNF180 and Septin9.

The present application also discloses a method for grading diseaseseverity in an individual, comprising determining the methylation levelwithin at least one target region of RNF180 gene or fragment thereof ina biological sample isolated from the individual, and grading diseaseseverity by the detection result of the methylation of RNF180.Preferably, the method further comprises determining the methylationlevel within at least one target region of Septin9 gene or fragmentthereof in a biological sample isolated from the individual, and gradingthe disease severity by combining the detection results of methylationof RNF180 and Septin9.

Typically, the method according to the present application furthercomprises the step of using a reagent to convert 5-unmethylated cytosinebase of a gene to uracil or other base that is detectably different fromcytosine in terms of hybridization performance.

Bisulfite modification of DNA is a known tool used to assess CpGmethylation status. 5-methylcytosine is the most common covalent basemodification in a DNA of a eukaryotic cell, and plays a role, forexample, in transcriptional regulation, in genetic imprinting, and intumorigenesis. Therefore, the identification of 5-methylcytosine as acomponent of genetic information is of considerable significance.However, 5-methylcytosine cannot be identified by sequencing, because5-methylcytosine has the same base pairing behavior as cytosine.Moreover, the epigenetic information carried by 5-methylcytosine iscompletely lost during, e.g., PCR amplification.

The most frequently used method for analyzing DNA for the presence of5-methylcytosine is based upon the specific reaction of bisulfite withcytosine whereby upon subsequent alkaline hydrolysis, cytosine isconverted to uracil which corresponds to thymine in its base pairingbehavior. However, it is noted that 5-methylcytosine remains unmodifiedunder these conditions. Consequently, an original DNA is converted insuch a manner that methylcytosine, which originally could not bedistinguished from cytosine by its hybridization behavior, can now bedetected as the only remaining cytosine using conventional and knownmolecular biological techniques, for example, by amplification andhybridization. All of these techniques are based on differential basepairing properties, which can now be fully exploited.

Therefore, typically, the present invention provides use of bisulfitetechnique, in combination with one or more methylation assays, fordetermination of the methylation status of CpG dinucleotide sequenceswithin Septin9 and RNF180 gene sequences. Genomic CpG dinucleotides canbe methylated or unmethylated (alternatively known as up- anddown-methylated respectively). However, the methods of the presentinvention are suitable for the analysis of a heterogeneous biologicalsample, e.g., a low concentration of tumor cells within blood or stool.Accordingly, when analyzing the methylation level at a CpG positionwithin such a sample, a person skilled in the art may use a quantitativeassay for determining the methylation level (e.g., percent, fraction,ratio, proportion or degree) at a particular CpG position other than amethylation status. Accordingly, the term methylation state ormethylation status should also be taken as a value reflecting themethylation level at a CpG position. Unless specifically stated, theterm “hypermethylated” or “upmethylated” shall be taken as a methylationlevel above a specified critical value, wherein the critical value maybe a value representing the average or median methylation level for agiven population, or is preferably an optimized critical level. The“critical value” is also referred herein as a “threshold value”. In thecontext of the present invention, the term “methylated”,“hypermethylated” or “upmethylated” shall be taken to include amethylation level above the critical value zero (0) % (or an equivalentthereof) for all CpG positions within and associated with (e.g. in apromoter or a regulatory region) the genes or genomic sequence selectedfrom the group consisting of the above Septin 9 and RNF180 genesequences.

In certain embodiments, the method of the present applicationspecifically comprises: contacting Septin9 and RNF180 genes or fragmentsthereof treated by the reagent with an amplification enzyme and primerssuch that the treated genes or fragments are amplified to produceamplification products or not amplified; detecting the amplificationproducts with probes; and determining the methylation level of at leastone CpG dinucleotide of the DNA sequences of Septin9 and RNF180 genesbased on the presence or absence of the amplification products.

Also, the contact or amplification typically comprises applying at leastone method selected from the group consisting of: use of a thermostableDNA polymerase as the amplification enzyme; use of a polymerase lacking5′-3′ exonuclease activity; use of a polymerase chain reaction (PCR);and generation of a nucleic acid molecule with an amplification productwith a detectable label.

That is, the methylation level is preferably determined by a PCR method,and methods such as “fluorescence-based real-time PCR technique” (Eadset al., Cancer Res. 59: 2302-2306, 1999), Ms-SNuPE TM(Methylation-sensitive Single Nucleotide Primer Extension) reaction(Gonzalgo & Jones, Nucleic Acids Res. 25: 2529-2531, 1997),methylation-specific PCR (“MSP”; Herman et al., Proc. Natl. Acad. Sci.USA 93: 9821-9826, 1996; U.S. Pat. No. 5,786,146), and methylated CpGisland amplification (“MCA”; Toyota et al., Cancer Res. 59: 2307-12,1999) are used to detect the methylation level of at least one CpGdinucleotide of the DNA sequences of Septin9 and RNF180 gene.

Among these, “fluorescence-based real-time PCR” is a high-throughputquantitative methylation assay that utilizes fluorescence-basedreal-time PCR (TaqMan) technology, and requires no further manipulationsafter the PCR step (Eads et al., Cancer Res. 59: 2302-2306, 1999).Briefly, “fluorescence-based real-time PCR” process begins with a mixedsample of genomic DNA that is converted, in a sodium bisulfite reaction,to a mixed pool of methylation-dependent sequence differences accordingto the standard procedures (the bisulfite process converts unmethylatedcytosine residues to uracil). Fluorescence-based PCR is then performedin a “biased” (with the PCR primers that overlap known CpGdinucleotides) reaction. Sequence discrimination can occur both at thelevel of the amplification process and at the level of the fluorescencedetection process.

“Fluorescence-based real-time PCR” assay may be used as a quantitativetest for methylation pattern in a genomic DNA sample, wherein sequencediscrimination occurs at the level of probe hybridization. In thisquantitative manner, the PCR reaction provides for a methylationspecific amplification in the presence of a fluorescent probe thatoverlaps a particular putative methylation site. An unbiased control forthe amount of input DNA is provided by a reaction in which neither theprimers, nor the probes overlie any CpG dinucleotides.“Fluorescence-based real-time PCR” method can be used with any suitableprobes, such as “TaqMan_”, “Lightcycler_”, etc.

TaqMan probe is dual-labeled with fluorescent “reporter” and “quencher”molecules, and is designed to be specific for a region having arelatively high GC content so that it melts out in the PCR cycle at anabout 10° C. higher temperature than the forward or reverse primer. Thisallows TaqMan_ probe to remain fully hybridized during the PCRannealing/extension step. As a Taq polymerase enzymatically synthesizesa new strand during PCR, it will eventually reach the annealed TaqMan_probe. The Taq polymerase 5′ to 3′ endonuclease activity will thendisplace the TaqMan_ probe by digesting it to release the fluorescentreporter molecule for quantitative detection of its now unquenchedsignal using a real-time fluorescent detection system.

A typical reagent (e.g., can be found in a kit basedon“fluorescence-based real-time PCR”) for “fluorescence-based real-timePCR” assay may include, but are not limited to: PCR primers for aparticular gene (or DNA sequence or CpG island treated by bisulfite);TaqMan_ or Lightcycler_(—) probes; optimized PCR buffers anddeoxynucleotides; and a Taq polymerase.

However, specifically, in a preferred embodiment, the method comprisesthe following steps:

In a first step, a tissue sample to be analyzed is obtained. The sourcemay be any suitable sources, such as cell lines, histological sections,tissue biopsies, paraffin-embedded tissues, body fluids, stool, coloniceffluent, urine, plasma, serum, whole blood, isolated blood cells, cellsisolated from blood, and any possible combination thereof. Preferably,the source of DNA is stool or body fluids selected from the groupconsisting of colonic effluent, urine, plasma, serum, whole blood,isolated blood cells, and cells isolated from blood.

Genomic DNA is then isolated from the sample by any standard means inthe prior art, including use of a commercially available kit. Briefly,where the DNA of interest is encapsulated in cellular membrane, thebiological sample must be disrupted and lysed by enzymatic, chemical ormechanical means. Then proteins and other contaminants can be removede.g., by digestion with proteinase K. The genomic DNA is then recoveredfrom the solution. This may be carried out by means of a variety ofmethods including salting out, organic extraction, or binding of the DNAto a solid phase support. The choice of method will be affected byseveral factors including time, expense and quantity of DNA as required.

When the sample DNA is not enclosed in cell membrane (e.g. circulatingDNA from a blood sample), the standard methods in the art for isolationand/or purification of DNA may be employed. Such methods include use ofa protein degenerating reagent, e.g., chaotropic salt, such as guanidinehydrochloride or urea; or a detergent, e.g. sodium dodecyl sulphate(SDS), cyanogen bromide. Alternative methods include, but are notlimited to, ethanol precipitation or propanol precipitation, vacuumconcentration by means of centrifuge amongst others. A person skilled inthe art may also make use of devices, such as filter devices, e.g.,ultrafiltration, silica surfaces or membranes, magnetic particles,polystyrol particles, polystyrol surfaces, positively charged surfaces,and positively charged membranes, charged membranes, charged surfaces,charged switch membranes, charged switch surfaces.

Once the nucleic acids have been extracted, the genomic double strandedDNA is used in the analysis.

In the second step of the method, the genomic DNA sample is treated insuch a manner that 5′-unmethylated cytosine base is converted to uracil,thymine, or another base which is different from cytosine in terms ofhybridization behaviour. This will be understood as “pre-treatmen” or“treatment” herein.

This is preferably achieved by means of treatment with a bisulfitereagent. The term “bisulfite reagent” refers to a reagent includingbisulfite, disulfite, hydrogen sulfite, or combination thereof, which isuseful as disclosed herein to distinguish between methylated andunmethylated CpG dinucleotide sequences. The treatment is known in theart (e.g., PCT/EP2004/011715, which is incorporated by reference in itsentirety). It is preferred that the bisulfite treatment is conducted inthe presence of a denaturing solvent, such as but not limited to,n-alkylene glycol, particularly diethylene glycol dimethyl ether (DME),or in the presence of dioxane or dioxane derivatives. In a preferredembodiment, the denaturing solvent is used in a concentration between 1%and 35% (v/v). It is also preferred that the bisulfite reaction iscarried out in the presence of a scavenger, such as but not limited to,a chromane derivative, e.g., 6-hydroxy-2,5,7,8-tetramethylchromane2-carboxylic acid or trihydroxybenzoic acid, and derivates thereof,e.g., gallic acid (see PCT/EP2004/011715, which is incorporated byreference in its entirety). The bisulfite conversion is preferablycarried out at a reaction temperature between 30° C. and 70° C., wherebythe temperature is increased to over 85° C. in a short period of timeduring the reaction (see PCT/EP2004/011715, which is incorporated byreference in its entirety). The bisulfite treated DNA is preferablypurified prior to the quantification. This may be conducted by any meansknown in the art, such as but not limited to, ultrafiltration,preferably carried out by means of Microcon^(λ)™ column (manufactured byMillipore^(λ)™).

In the third step of the method, the fragments of the treated DNA areamplified using the primer oligonucleotides according to the presentinvention and an amplification enzyme. The amplification of several DNAfragments can be carried out simultaneously in one same reaction vessel.Typically, the amplification reaction is carried out by a polymerasechain reaction (PCR). Preferably, the amplification products are 100 to2,000 base pairs in length.

The methylation of Septin9 gene or fragment thereof is detected by e.g.the following primers and probe for Septin9:

Primer:  SEQ ID No: 14 GTAGTAGTTAGTTTAGTATTTATTTT Primer:  SEQ ID No: 15CCCACCAACCATCATAT Probe:  SEQ ID No: 16 GAACCCCGCGATCAACGCG

The methylation of RNF180 gene or fragment thereof is detected by theprimers and probes for RNF180 screened by the above-described screeningmethods. For example, in a preferred embodiment, any one of combinations1, 2, 3 and 4 of primers and probes described above can be used.

The fragments obtained by the amplification can carry a label which isdirectly or indirectly detectable. Preferably, the label is in the formof a fluorescence label, a radionuclide, or an attachable moleculefragment.

In the fourth step of the method, the amplification products obtained inthe third step of the method are analysed in order to determine themethylation status of the CpG dinucleotides prior to the treatment.

In the fourth step, the detection of the amplification products isconducted with probes for a real-time detection. In the presentinvention, the real-time PCR detection can be performed on a variety ofcommercial real-time PCR apparatus according to the standard operationsin the prior art. The real-time PCR detection is performed on a LifeTechnologies instrument (7500Fast) according to certain specificembodiments. The PCR reaction mixture consists of thebisulfite-converted DNA templates 25-40 ng and 300-600 nM primers,150-300 nM probes, 1UTaq polymerase, 50-400 uM each dNTPs, 1 to 10 mMMgCl2 and 2×PCR buffer with a final volume of 2 ul to 100 ul, which iskept at 85 to 99° C. for 3-60 minutes to amplify samples in pre-cycling,followed by 35 to 55 cycles of annealing at 50 to 72° C. for 1 to 30seconds, annealing at 45 to 80° C. for 5 to 90 seconds, and denaturingat 85 to 99° C. for 5 to 90 seconds.

Only by the amplification observed on the methylated RNF180 gene andSeptin9 gene fragments, the gene fragments are detected with the probesspecific for the RNF180 and Septin9 promoter regions contained5-methylcytosine. Furthermore, in certain specific embodiments, β-actinis used as an internal reference in PCR, the β-actin amplicon is createdby using the primers complementary to the β-actin sequence, and theβ-actin amplicon is detected with the particular probes. Each sample issubjected to at least one real-time PCR, and in some embodiments, twodetections based on real-time PCR are performed.

In the fifth step of the method, the methylation status of at least oneCpG dinucleotide in the DNA sequence of Septin9 and RNF180 genes arepresented respectively, which is determined by the cycle threshold Ctvalue of the polymerase chain reaction, and then the following steps arefurther included: A) comparing the Ct values of PCR corresponding toSeptin9 in the tested sample to the predetermined cut value (i.e., thecritical Ct value) of Septin9 to determine whether the analysis resultbased on Septin9 gene is positive; B) comparing Ct values of PCRcorresponding to RNF180 in the tested sample to a predetermined cutvalue of RNF180 to determine whether the analysis result based on theRNF180 gene represents normal, gastritis or gastric cancer (positive);C) combining the results of steps A) and B) to determine whether thefinal analysis result of the sample represents normal, gastritis, orgastric cancer (positive).

According to the specific embodiments of the present application, thecritical Ct values for gastric cancer, gastritis, and normal withrespect to Septin9 and RNF180 are determined based on the mean Ct valuesof Septin9 and RNF180 of a number of gastric cancer samples and normalsamples. In certain preferred embodiments, the kit for measuring themethylation status of at least one CpG dinucleotide of the DNA sequenceof Septin9 gene was purchased from Epigenomics AG (Germany), so thatcritical Ct value of Septin 9 can be determined as 45 according to theinstructions of Epigenomics AG. And the critical Ct value of RNF180 isdetermined based on the mean Ct value of RNF180 in a certain number ofgastric cancer, gastritis and normal samples. And the critical Ct valueof RNF180 is also related to the actual sensitivity as required, and thehigher the sensitivity requirement is, the greater the selected criticalCt value is.

Furthermore, the present application allows the analysis of Ct valueusing different methodologies. For example, ΔCt or dCT is used, and theCt of actin is takes as the internal control of the PCR, and the dCTvalue of Septin 9 is obtained by substracting the Ct of actin from theCt of Septin 9. Similarly, the dCT value of RNF 180 is obtained bysubstracting the Ct of actin from the Ct of RNF 180. Accordingly, if ΔCtor dCT is used as the detection standard, in the fifth step of themethod, the methylation status of at least one CpG dinucleotide withinthe DNA sequences of Septin9 and RNF180 genes is presented respectively,which is determined by the cycle threshold Ct value of the polymerasechain reaction, and then the following steps are further included: A)comparing the ΔCt values of PCR corresponding to Septin9 in the testedsample to the pre-determined Δcut value (i.e., the critical Ct value) ofSeptin9 to determine whether the analysis result based on Septin9 geneis positive; B) comparing the ΔCt values of PCR corresponding to RNF180in the tested sample to the pre-determined Δcut value of RNF180 todetermine whether the analysis result based on RNF180 gene representsnormal, gastritis or gastric cancer (positive); C) combing the resultsof steps A) and B) to determine whether the final analysis result of thesamples represents normal, gastritis or gastric cancer (positive).

In summary, the present application improves the sensitivity andspecificity of cancer detection, especially those of gastric cancerdetection, by the above-described composition, nucleic acid sequences,kit, use thereof, and above-described detection method, which combinethe nucleic acid sequences for detecting the methylation of Septin9 andRNF180 genes or fragments thereof respectively, and thus ensures theaccuracy and reliability of the detection results; and has achievedgrading of gastritis and gastric cancer by combining the two biomarkersSeptin9 and RNF180, so that both the sensitivity and specificity ofdisease grading are improved.

The specific examples will be described in detail below.

EXAMPLES Example 1: Screening of Primers and Probes

According to certain specific embodiments, the kit for measuring themethylation status of at least one CpG dinucleotide of the DNA sequenceof Septin9 gene was purchased from Epigenomics AG (Germany). Therefore,the primers and probes for the experiment for Septin9 gene were obtaineddirectly according to the instructions of the kit. For RNF180 gene,multiple sets of combinations of probes and primers can be designed, andeach combination of the probes and primers may be different in theperformance. The probes and primers were screened in the followingexamples.

In this example, the primers and probes for RNF180 were first screenedwith an artificially methylated template and an unmethylated template.The example includes the following steps:

First, various primers and probes for RNF180 were designed, providedthat they are capable of being equivalent to, complementary to, orhybridize under moderately stringent or stringent conditions to at least15 nucleotides selected from the group consisting of SEQ ID Nos: 10 to12, or complementary sequence thereof.

The PCR amplifications were then carried out with the differentcombinations of probes and primers using an artificially methylatedoligonucleotide sequence and an artificially unmethylatedoligonucleotide sequence as templates. The conditions for the PCRamplifications employed in the experiment were as follows: the real-timePCR was performed on a Life Technologies instrument (7500Fast). The PCRreaction mixture consisted of 35 ng bisulfite-converted DNA templatesand 450 nM primers, 225 nM probes, 1UTaq polymerases, 200 um each dNTPs,4.5 mM MgCl2 and 2×PCR buffer with a final volume of 30 ul, which waskept at 94° C. for 20 minutes to amplify samples in precycling, followedby 45 cycles of annealing at 62° C. for 5 seconds, annealing at 55.5° C.for 35 seconds, and denaturing at 93° C. for 30 seconds.

Finally, the four sets of suitable primers and probes were screenedbased on the PCR experiment results:

Primers and probes combination 1 (amplicon 66 bp, −43 bp to +23b fromthe transcription initiation site)

(180F7) SEQ ID No: 1 5′-GTTCGAGGTCGCGGGGTC-3′ (180P7) SEQ ID No: 25′-CAL Fluor Red-AACGCTCGAACTATACCTACAACCCC- BHQ2-3′ (180R7)SEQ ID No: 3 5′-ACAAAAACCAAACCCCGCG-3′

Primers and probes combination 2 (amplicon 86 bp, +5 bp to +91 bp withrespect to the transcription initiation site)

(180F24) SEQ ID No: 4 5′-GCGGGGTTTGGTTTTTGT-3′ (180P2) SEQ ID No: 55′-CAL Fluor Red-CCGACGACGACGATACCG-BHQ2- 3′ (180R2) SEQ ID No: 65′-ACAACCAAACTCTAAAAACTCG-3′

Primers and probes combination 3 (amplicon 130 bp, +5 bp to +135 bp withrespect to the transcription initiation site)

(180F24) SEQ ID No: 4 5′-GCGGGGTTTGGTTTTTGT-3′ (180P14) SEQ ID No: 75′-CAL Fluor Red-CGTCGGAGTCGTAGCGAGTTT-BHQ2-3′ (180R135) SEQ ID No: 85′-AAAACCTCCAACTTCACACCC-3′

Primers and probes combination 4 (amplicon 91 bp, +5 bp to +96 bp withrespect to the transcription initiation site)

(180F24) SEQ ID No: 4 5′-GCGGGGTTTGGTTTTTGT-3′ (180P14) SEQ ID No: 75′-CAL Fluor Red-CGTCGGAGTCGTAGCGAGTTT-BHQ2-3′ (180R14) SEQ ID No: 95′-CGCCAACAACCAAACTCTAA-3′

Conclusion: there was amplification based on the artificially methylatedoligonucleotide template and no amplification based on the artificiallyunmethylated oligonucleotide template, and it indicates that the primersand probes are designed correctly. The four sets of primers and probesare capable of distinguishing the methylated template from theunmethylated template, all of which could be used as the primers andprobes in the experiment for RNF180. Although the effects of thedifferent combinations of probes and primers are different, all theabove four sets of probes are suitable as the primers and probes in theexperiment for RNF180.

Next, the primers and probes for RNF180 were further screened withcancer and normal DNA as templates.

The samples were obtained from 4 cases of gastric cancer (S1-S4), 2cases of colon cancer (C1-C2), 2 cases of lung cancer (L1-L2), 1 case ofblood cancer (Jurkat) and 1 case of normal people (WBC). The GenomicDNAs were extracted from 4 cases of gastric cancer, 2 cases of coloncancer, 2 cases of lung cancer, 1 case of blood cancer and 1 case ofnormal people. The genomic DNA of Jurkat cells was used as a positivecontrol, and normal DNA as a negative control. All cancer samples wereobtained from BIOCHAIN INC. The normal people sample was obtained fromBioReclamation IVT Inc. The extraction of DNA can be carried out usingany standard means in the prior art, and in particular, in this example,all the DNAs of human samples were extracted by the EPi proColon PlasmaQuick Kit of Epigenomics AG.

The genomic DNA sample was then pretreated such that 5′-unmethylatedcytosine base was converted to uracil, thymine, or another base which isdifferent from cytosine in terms of hybridization behaviour. In theexample, this pretreatment was achieved by treatment with a bisulfitereagent. The DNA modification with bisulfite was carried out by the EPiproColon Plasma Quick Kit.

Next, the four sets of primers and probes for RNF180 described abovewere added to the pre-treated genomic DNA samples of 4 cases of gastriccancer, 2 cases of colon cancer, 2 cases of lung cancer, 1 case of bloodcancer and 1 case of normal person to carry out 4 groups of PCRexperiments for RNF 180, and the primers and probes for Septin9 wereadded for multiple detection of the PCR experiments for RNF180 andSeptin9. The PCR reagents for Septin9 were purchased from EpigenomicsAG. The Real-time PCR was performed with the bisulfite-converted DNAs.

The PCR amplification conditions employed in the experiment were asfollows: the real-time PCR detection was performed on a LifeTechnologies instrument (7500Fast). The PCR reaction mixture consistedof the bisulfite-converted DNA templates 35 ng and 450 nM primers, 225nM probes, 1UTaq polymerases, 200 um each dNTPs, 4.5 mM MgCl2 and 2×PCRbuffer with a final volume of 30 ul, which was kept at 94° C. for 20minutes to amplify samples in precycling, followed by 45 cycles ofannealing at 62° C. for 5 seconds, annealing at 55.5° C. for 35 seconds,and denaturing at 93° C. for 30 seconds.

Finally, the Ct values of real-time PCR for RNF180 gene in the genomicDNA samples of 4 cases of gastric cancer, 2 cases of colon cancer, 2cases of lung cancer, 1 case of blood cancer and 1 case of normal personwere determined, and the Ct values of real-time PCR for Setptin9 gene inthe genomic DNA samples of 4 cases of gastric cancer, 2 cases of coloncancer, 2 cases of lung cancer, 1 case of blood cancer and 1 case ofnormal people were determined (as shown in FIG. 1). FIG. 1 shows that inthe multiple detection of the methylated DNAs of Septin9 and RNF180,none of the four sets of primers and probes for RNF180 affects thedetection of Septin9. The abscissa SC_set 1-4 represents the sets ofprimers and probes 1-4, the bars in each set represent 51, S2, S3, S4,C1, C2, L1, L2, Jurkat and WBC from left to right respectively, and theordinate is the Ct value of Septin9, wherein S1-S4 represent 4 cases ofgastric cancer, C1-C2 represent 2 cases of colon cancer, L1-L2 represent2 cases of lung cancer, Jurkat represents 1 case of blood cancer(positive control), and WBC represents 1 case of normal people (negativecontrol).

It can be seen from the Ct value of the real-time PCR for RNF180 genethat the amplification of RNF180 is high in cancerous DNA, and low innormal DNA. The specificity of primers and probes for RNF180 candistinguish from cancerous DNA and normal DNA. The differentcombinations of primers and probes for RNF180 achieve different effect.The amplification of RNF180 is very high in the DNA of blood cancerJurkat cells.

As shown in FIG. 1, it can be seen from the Ct value of the real-timePCR of Setptin9 gene that the primers and probes for RNF180 do notaffect the detection of Septin9 in the multiple detection. The primersand probes for Septin9 can distinguish from cancerous DNA and normalDNA, without being affected by RNF180. The different combinations ofprimers and probes for RNF180 do not affect the detection of Septin9.There is very high amplification of RNF180, but no amplification ofSeptin9 in the DNA of blood cancer Jurkat cells. The methylation ofRNF180 and Septin9 is similar in some cancers, but different in others.

According to the above example, all of the four sets of probes andprimers for RNF180 designed by the present application can distinguishcancerous DNA from normal DNA. Further, in the following examples, theabove-mentioned probes and primers were used to further compare andanalyze the methylation level of the DNA in the biological samples ofnormal people, patients with gastritis, and patients with gastriccancer. In particular, in order to enable the grading of diseaseseverity using the result of a real-time PCR, a critical value of Ct,i.e. a cut value of a real-time PCR, is first determined by analyzing acertain amount of samples, and the cut value serves to grade diseaseseverity according to the different requirements on sensitivities andspecificities. In the following examples, the corresponding Ct valueswhich reflect the levels of methylated DNA of Septin9 and RNF180 weredetermined in the biological samples of the patients with gastriccancer, patients with gastritis, and normal people, thereby providing ajudgmental critical value, i.e. the critical cut value, for gradingdisease severity (gastric cancer, gastritis, and normal).

Example 2: Preliminary Clinical Results of Multiple Detection ofMethylated DNA of RNF180 and Septin9 in Plasma from Patients withGastric Cancer

The samples were obtained from 10 patients with gastric cancer and 11healthy people. The Genomic DNAs were extracted from the 10 patientswith gastric cancer and 11 healthy people. All the cancer samples wereobtained from BIOCHAIN Inc. The normal people sample was obtained fromBioReclamation IVT Inc. The extraction of DNA can be carried out usingany standard means in the prior art, and in particular, in this example,the DNAs of all samples were extracted by the EPi proColon Plasma QuickKit of Epigenomics AG.

The genomic DNA samples were then pretreated such that 5′-unmethylatedcytosine base was converted to uracil, thymine, or another base which isdifferent from cytosine in terms of hybridization behaviour. In theexample, this pretreatment was achieved by treatment with a bisulfitereagent. The DNA modification with bisulfite was carried out by the EPiproColon Plasma Quick Kit.

Next, the second set of primers and probes for RNF180 described abovewas added to the pre-treated genomic DNA samples of 10 patients withgastric cancer and 11 healthy people to carry out the PCR experiment forRNF 180, and the primers and probes for Septin9 were added for multipledetection of the PCR experiments for RNF180 and Septin9. The PCRreagents for Septin9 were purchased from Epigenomics AG. The Real-timePCR was performed with the bisulfite-converted DNAs.

The PCR amplification conditions employed in the experiment were asfollows: the real-time PCR was performed on a Life Technologiesinstrument (7500Fast). The PCR reaction mixture consisted of thebisulfite-converted DNA templates 35 ng and 450 nM primers, 225 nMprobes, 1UTaq polymerases, 200 um each dNTPs, 4.5 mM MgCl2 and 2×PCRbuffer with a final volume of 30 ul, which was kept at 94° C. for 20minutes to amplify samples in precycling, followed by 45 cycles ofannealing at 62° C. for 5 seconds, annealing at 55.5° C. for 35 seconds,denaturing at 93° C. for 30 seconds.

Finally, the Ct values of real-time PCR for RNF180 gene in the genomicDNA samples from 10 patients with gastric cancer and 11 healthy peoplewere determined, and the Ct values of real-time PCR for Setptin9 gene inthe genomic DNA samples from 10 patients with gastric cancer and 11healthy people were determined respectively, as shown in the followingtable 1-3.

TABLE 1 multiple detection of methylated DNA of Septin9 and RNF180 in 10patients with gastric cancer Ct Ct Cancer samples RNF180 Septin9Correlation Diagnosis 6/Asian/male/63 / 38.19 complementary Positive35/Asian/male/58 37.62 / complementary Positive 36/Asian/female/53 34.4638.34 Positive 39/Asian/female/58 31.84 34.51 Positive 41/Asian/male/5333.28 38.67 Positive 42/Asian/female/58 36.96 / complementary Positive43/Asian/female/56 32.90 36.03 Positive 59/Asian/male/57 34.56 38.00Positive 61/Asian/male/69 34.71 / complementary Positive68/Asian/female/49 35.15 No Ct complementary positive Positive rate 90%70% Positive

TABLE 2 multi-detection of methylated DNA of Septin9 and RNF180 in 6healthy people Plasma from healthy people Ct RNF180 Ct Septin9H1/Asian/male/30 / / H2/Asian/female/27 36.45 / H3/Asian/male/24 / /H4/Asian/female/45 / / H5/Asian/male/35 / / H6/Asian/male and female/32/ / Specificity 83% 100%

TABLE 3 multi-detection of methylated DNA of Septin9 and RNF180 in 5healthy people RNF180 Septin9 Sample Ct mean value Ct SD Ct mean valueCt SD #1: Spain/male/30 / / / / #2: Black/male/34 / / / / #3:Black/female/19 / / / / #4: Black/female/31 / / / / #5: Black/female/42/ / / / RC1 28.65 0.01 38.14 0.60 RC2 28.42 0.01 38.46 1.71 Gastriccancer #3 29.93 0.03 29.86 0.15 Water / / / /

Both RC1 and RC2 are the references. A Ct value of 45 is taken as acritical value for the PCR of RNF180 and Septin9, and the Ct value above45 represents normal, and the Ct value below 45 represents positive forcancer. “/” indicates the Ct value is above 45.

In the multiple detection of the methylated DNA of Septin9 and RNF180,the Ct value below 45 represents positive for gastric cancer. Thepositive results of the detection of methylated DNA of Septin9 andRNF180 are combined, and complementary to each other. 10 patients withgastric cancer are diagnosed as positive. The sensitivity of gastriccancer reaches 100%. In 11 healthy people, 10 are diagnosed as negativeand the specificity reaches 91%.

The Ct values of PCR of RNF180 and Septin9 in 10 gastric cancer patientsin Table 1 are shown in the form of a histogram, as shown in FIG. 2. Theabscissa in FIG. 2 is 10 cases of gastric cancer and the ordinate is theCt values of Septin9 and RNF180.

The positive results of methylated DNA detection of Septin9 and RNF180are combined, that is, the positive results of methylated DNA detectionof Septin9 and RNF180 are complementary to each other, and thesensitivity of gastric cancer reaches 100%. The positive results ofSeptin9 and RNF180 are complementary in the diagnosis of gastric cancer.

Example 3: Grading of Gastric Cancer, Gastritis, and Normal People byMultiple Detection of Methylated DNA of RNF180 and Septin9

The example comprises the following steps:

First, the plasma was obtained from 42 patients with gastric cancer, 14patients with gastritis, and 11 normal people. The Genomic DNAs wereextracted from the patients with gastric cancer, patients withgastritis, and normal people. All the cancer samples were obtained fromBIOCHAIN Inc. The normal people samples were obtained fromBioReclamation IVT Inc. The Extraction of DNA can be carried out usingany standard means in the prior art, and in particular, in this example,the DNAs of all samples were extracted by the EPi proColon Plasma QuickKit of Epigenomics AG.

The genomic DNA samples were then pretreated such that 5′-unmethylatedcytosine base was converted to uracil, thymine, or another base which isdifferent from cytosine in terms of hybridization behaviour. In theexample, this pretreatment was achieved by treatment with a bisulfitereagent. The DNA modification with bisulfite was carried out by the EPiproColon Plasma Quick Kit.

Next, the second set of primers and probes for RNF180 described abovewere added to the pre-treated genomic DNA samples of 42 patients withgastric cancer, 14 patients with gastritis, and 11 normal people tocarry out the PCR experiments for RNF 180, and the primers and probesfor Septin9 were added for multiple detection of the PCR experiments forRNF180 and Septin9. The PCR reagents for Septin9 were purchased fromEpigenomics AG. The Real-time PCR was performed with thebisulfite-converted DNAs.

The PCR amplification conditions employed in the experiments were asfollows: the real-time PCR detection was performed on a LifeTechnologies instrument (7500Fast). The PCR reaction mixture consistedof the bisulfite-converted DNA templates 35 ng and 450 nM primers, 225nM probes, 1UTaq polymerases, 200 um each dNTPs, 4.5 mM MgCl2 and 2×PCRbuffer with a final volume of 30 ul, which was kept at 94° C. for 20minutes to amplify samples in precycling, followed by 45 cycles ofannealing at 62° C. for 5 seconds, annealing at 55.5° C. for 35 seconds,denaturing at 93° C. for 30 seconds.

Finally, the Ct values of real-time PCR for RNF180 gene in the genomicDNA samples of 42 patients with gastric cancer, 14 patients withgastritis, and 11 normal people were determined, and the Ct values ofreal-time PCR for Setptin9 gene in the genomic DNA samples of 42patients with gastric cancer, 14 patients with gastritis, and 11 normalpeople were determined, respectively. And a statistical analysis wasperformed according to the determined Ct values. FIGS. 3 and 4 are thehistogram and scatter diagram of the mean Ct values of multipledetection of methylated DNA of Septin9 and RNF180 in gastric cancer,gastritis, and normal people. The abscissa represents from the left tothe right the mean Ct values of the methylation level of RNF180 gene in11 normal people, the mean Ct values of the methylation level of RNF180gene in 14 patients with gastritis, the mean Ct values of methylationlevel of RNF180 gene in 42 patients with gastric cancer, the mean Ctvalues of methylation level of Setptin9 gene in 11 normal people, themean Ct values of methylation level of Setptin9 gene in 14 patients withgastritis, and the mean Ct values of methylation level of Setptin9 genein 42 patients with gastric cancer, respectively. * represents asignificant difference, ** represents a very significant difference, and*** represents an extremely significant difference.

It can be seen from the above figures that in the detection of RNF180,there is a very significant difference of Ct values between gastriccancer and gastritis, a significant difference between normal people andgastritis, and an extremely significant difference between normal peopleand gastric cancer, thus providing a critical value for judgement, i.e.the cut value for grading disease severity (gastric cancer, gastritis,and normal). It also indicates that the disease severity can be gradedby the detection result of methylation of RNF180, for example, gastriccancer, gastritis, and normal people can be graded. In the detection ofSeptin9, there is a significant difference of Ct value between gastriccancer and gastritis, and a significant difference between normal peopleand gastric cancer.

In this example, the sensitivity of the gastric cancer detection is83.3%, the sensitivity of the gastritis detection is 64.3%, and thespecificity of completely normal is 90.9% when the Ct value of 45 istaken as the critical value for Septin9 and the Ct value of 45 is takenas the critical value for RNF180. The sensitivity of gastric cancer is78.6%, the sensitivity of gastritis is 28.3%, and the specificity ofcompletely normal is 90.9% when the Ct value of 45 is taken as thecritical value for Septin9, a Ct value of RNF180 below 39 presentsgastric cancer, and a Ct value of RNF180 of 39-45 presents gastritis.

The above experimental results indicate that disease severity can begraded by the multiple detection of methylated DNA of RNF180 and Septin9of the present invention, for example, gastric cancer, gastritis, andnormal people can be graded.

Example 4: Grading of Normal People, Superficial, AtrophicGastritis/Gastritis with Intestinal Metaplasia, Gastric Cancer Stages I,II, III and IV Based on Multiple Detection of Methylated DNA of RNF180and Septin9 (RS19)

The samples were obtained from 15 normal people, 31 cases of superficialgastritis, 6 cases of atrophic gastritis/gastritis with intestinalmetaplasia, 19 cases of gastric cancer stage I, 20 cases of gastriccancer stage II, 22 cases of gastric cancer stage III and 10 cases ofgastric cancer stage IV. The Genomic DNA was extracted from each sample.The Extraction of DNA can be carried out with any standard means knownin the prior art, and particularly, in this example, the DNAs of allsamples were extracted by the EPi proColon Plasma Quick Kit ofEpigenomics AG.

The genomic DNA samples were then pretreated such that 5′-unmethylatedcytosine base was converted to uracil, thymine, or another base which isdifferent from cytosine in terms of hybridization behaviour. In theexample, the pretreatment was achieved by a bisulfite reagent. The DNAmodification with the bisulfite was carried out by the EPi proColonPlasma Quick Kit.

Next, the second set of primers and probes for RNF180 described abovewere added to the pre-treated genomic DNA samples of 15 normal people,31 cases of superficial gastritis, 6 cases of atrophicgastritis/gastritis with intestinal metaplasia, 19 cases of gastriccancer stage I, 20 cases of gastric cancer stage II, 22 cases of gastriccancer stage III and 10 cases of gastric cancer stage IV so as to carryout the PCR experiment for RNF 180, and the primers and probes forSeptin9 and β-actin were added for the multiple detection of the PCRexperiments for RNF180, Septin9 and β-actin. The PCR reagents forSeptin9 and β-actin were purchased from Epigenomics AG. The Real-timePCR was performed with the DNAs converted by the bisulfite.

Wherein the PCR amplification conditions employed in the experiment wereas follows: the real-time PCR detection was performed on a LifeTechnologies instrument (7500Fast). The PCR reaction mixture consistedof 35 ng DNA templates converted by the bisulfite and 450 nM primers,225 nM probes, 1UTaq polymerases, 200 um each dNTPs, 4.5 mM MgCl2 and2×PCR buffer with a final volume of 30 ul, which was kept at 94° C. for20 minutes to amplify samples in precycling, followed by 45 cycles ofannealing at 62° C. for 5 seconds, annealing at 55.5° C. for 35 seconds,denaturing at 93° C. for 30 seconds.

Finally, the Ct values of the real-time PCR for Setptin9 gene in thegenomic DNA samples of 15 normal people, 31 cases of superficialgastritis, 6 cases of atrophic gastritis/gastritis with intestinalmetaplasia, 19 cases of gastric cancer stage I, 20 cases of gastriccancer stage II, 22 cases of gastric cancer stage III and 10 cases ofgastric cancer stage IV were determined. The Ct values of the real-timePCR of β-actin in the genomic DNA samples of 15 normal people, 31 casesof superficial gastritis, 6 cases of atrophic gastritis/gastritis withintestinal metaplasia, 19 cases of gastric cancer stage I, 20 cases ofgastric cancer stage II, 22 cases of gastric cancer stage III and 10cases of gastric cancer stage IV were also determined, as shown in FIG.5. It can be seen from FIG. 5 that the average Ct values of β-actin(ACTB) are very similar as the disease severity increases from normalpeople, superficial, atrophic gastritis/gastritis with intestinalmetaplasia to gastric cancer stages I, II, III, and IV. Thus, the Ctvalues of β-actin are constant. The Ct of β-actin can serve as aninternal reference for the PCR, i.e., an internal control.

The Ct values of the real-time PCR for RNF180 gene in the genomic DNAsamples of 15 normal people, 31 cases of superficial gastritis, 6 casesof atrophic gastritis/gastritis with intestinal metaplasia, 19 cases ofgastric cancer stage I, 20 cases of gastric cancer stage II, 22 cases ofgastric cancer stage III and 10 cases of gastric cancer stage IV aredetermined, as shown in FIG. 6. The dCT value of RNF180 is obtained bysubtracting the Ct of β-actin from the Ct of RNF180. In FIG. 6, theordinate is the dCt or ΔCt value of RNF 180.

Similarly, the dCT value of Septin9 is obtained by subtracting the Ct ofthe actin from the Ct of Septin 9. The lower the dCt value is, thehigher the methylation level is.

It can be seen from FIG. 6 that the dCt value of RNF180 is becominglower and the methylation level is becoming higher as the diseaseseverity increases from normal people, superficial gastritis, atrophicgastritis/gastritis with intestinal metaplasia to gastric cancer stagesI, II, III and IV.

The above experimental results further demonstrate that the Ct or dCtvalue can be used to not only grade normal people, gastritis, andgastric cancers, but also further grade gastritis, for example,gastritis can be graded into mild gastritis (including superficial) andsevere gastritis (including atrophic and with intestinal metaplasia).Furthermore, gastric cancer can be further graded based on the Ct or dCtvalue, for example, gastric cancer can be graded into gastric cancerstages I, II, III and IV.

Example 5: Determination of Positive Rates of Normal, SuperficialGastritis, Atrophic Gastritis/Gastritis with Intestinal Metaplasia,Gastric Cancer and Gastric Cancer Stages I, II, III and IV Based onMultiple Detection of Methylated DNA of Septin9 and RNF180 (RS19)

The samples were obtained from 15 normal people, 31 cases of superficialgastritis, 6 cases of atrophic gastritis/gastritis with intestinalmetaplasia (including 3 cases of atrophic gastritis and 3 cases ofgastritis with intestinal metaplasia), 74 cases of gastric cancer(including 19 cases of gastric cancer stage I, 20 cases of stage II, 22cases of stage III and 10 cases of stage IV). The genomic DNA wasextracted from each sample. The Extraction of the DNAs can be carriedout with any standard means in the prior art, and particularly, in thisexample, the DNAs of all samples were extracted by the EPi proColonPlasma Quick Kit of Epigenomics AG.

The genomic DNA samples were then pretreated such that 5′-unmethylatedcytosine base was converted to uracil, thymine, or another base which isdifferent from cytosine in terms of hybridization behaviour. In theexample, the pretreatment was achieved by treatment with a bisulfitereagent. The DNA modification with the bisulfite was carried out by theEPi proColon Plasma Quick Kit.

Next, the second set of primers and probes for RNF180 described abovewere added to the pre-treated genomic DNA samples of 15 normal people,31 cases of superficial gastritis, 6 cases of atrophicgastritis/gastritis with intestinal metaplasia, 74 cases of gastriccancer (including 19 cases of stage I, 20 cases of stage II, 22 cases ofstage III and 10 cases of stage IV) so as to carry out the PCRexperiment for RNF 180, and the primers and probes for Septin9 andβ-actin were added for multiple detection of the PCR experiments forRNF180, Septin9 and β-actin. The PCR reagents for Septin9 and β-actinwere purchased from Epigenomics AG. The Real-time PCR was performed onthe DNAs converted by bisulfite.

Wherein the PCR amplification conditions employed in the experiment wereas follows: the real-time PCR detections were performed on a LifeTechnologies instrument (7500Fast). The PCR reaction mixture consistedof 35 ng DNA templates converted by bisulfite, 450 nM primers, 225 nMprobes, 1UTaq polymerases, 200 um each dNTPs, 4.5 mM MgCl2 and 2×PCRbuffer with a final volume of 30 ul, which was kept at 94° C. for 20minutes to amplify samples in precycling, followed by 45 cycles ofannealing at 62° C. for 5 seconds, annealing at 55.5° C. for 35 seconds,denaturing at 93° C. for 30 seconds.

Finally, the Ct values of real-time PCR for Setptin9 gene in the genomicDNA samples of 15 normal people, 31 cases of superficial gastritis, 6cases of atrophic gastritis/gastritis with intestinal metaplasia, 74cases of gastric cancer (including 19 cases of stage I, 20 cases ofstage II, 22 cases of stage III and 10 cases of stage IV) weredetermined. The Ct values of real-time PCR for β-actin in the genomicDNA samples from 15 normal people, 31 cases of superficial gastritis, 6cases of atrophic gastritis/gastritis with intestinal metaplasia, 74cases of gastric cancer including 19 cases of stage I, 20 cases of stageII, 22 cases of stage III and 10 cases of stage IV were also determined.Further, the Ct values of real-time PCR for RNF180 gene in the genomicDNA samples of 15 normal people, 31 cases of superficial gastritis, 6cases of atrophic gastritis/gastritis with intestinal metaplasia, 74cases of gastric cancer (including 19 cases of stage I, 20 cases ofstage II, 22 cases of stage III and 10 cases of stage IV) weredetermined. The Ct for β-actin served as an internal control. The dCTvalue of RNF180 was obtained by subtracting the Ct of β-actin from theCt of RNF180. It was considered as positive for the diseases providedthat dCt<10.

FIG. 7A shows the comparation of positive rates of RS19 between normalpeople, superficial gastritis, atrophic gastritis/gastritis intestinalmetaplasia and gastric cancer. The ordinate represents positive rate,while the abscissa from left to right represents normal people,superficial gastritis, atrophic gastritis/gastritis with intestinalmetaplasia and gastric cancer, respectively. It can be seen from FIG. 7Athat the positive rate is becoming higher and higher as the diseaseseverity increases from normal people, superficial gastritis, atrophicgastritis/gastritis with intestinal metaplasia to gastric cancer.

FIG. 7B shows the comparation of positive rate for RS19 between gastriccancer stages I, II, III and IV. The ordinate represents the positiverate, while the abscissa from left to right represents gastric cancerstages I, II, III and IV, respectively. It can be seen from FIG. 7B thatthe positive rate is becoming higher and higher as the disease severityincreases from gastric cancer stages I, II, III to IV.

FIG. 7C shows the histogram of the average dCt values for RS19 of normalpeople, gastritis and gastric cancer, respectively. FIG. 7C furtherdemonstrates the results of Example 2. In the detection of RNF180, thereis a very significant difference of dCt value between gastric cancer andgastritis, a significant difference of dCt between normal people andgastritis, and an extremely significant difference of dCt between normalpeople and gastric cancer. Again, the results of the experiment confirmthat the disease severity can be graded by the detection result ofmethylation of RNF180, for example, gastric cancer, gastritis, andnormal people can be graded.

Furthermore, the methylation of RNF180 is of great significance in thechronic gastritis complicated with intestinal metaplasia. It isgenerally believed that chronic atrophic gastritis and chronic gastritiswith intestinal metaplasia are more prone to canceration. In threesamples which are diagnosed as chronic gastritis complicated withintestinal metaplasia according to pathological biopsies, the amounts ofRNF180 in blood increase, the Ct values decrease significantly, with theaverage Ct of 7.9 and the positive rate of 100% (3/3). However, thepositive rate of chronic atrophic gastritis is 33% (1/3), which isapproximate to that of chronic superficial gastritis of 27%, see Table4. It is estimated that the proportion of intestinal metaplasia in thepatients who have common chronic superficial gastritis and are positivefor RNF180 (27%), may increase if a biopsy is performed.

TABLE 4 dCT values of RNF180 in 3 cases of gastritis with intestinalmetaplasia and 3 cases of chronic atrophic gastritis Gastritis Case 1,Case 2, Case 3, Case 1, Case 2, Case 3, complicated complicatedcomplicated chronic chronic chronic with intestinal with intestinal withintestinal atrophic atrophic atrophic metaplasia metaplasia metaplasiagastritis gastritis gastritis dCT 5.5 7.5 10.6 6.2 22.2 23.1

Therefore, RNF180 can also be used to distinguish gastritis withintestinal metaplasia from common gastritis without intestinalmetaplasia, while the positive rate for RNF180 of gastritis withintestinal metaplasia is 100%, and the positive rate for RNF180 ofcommon gastritis is about 27%.

Example 6: Improvement of Combination of Septin9 and RNF180 onSpecificity and Sensitivity of Gastric Cancer Detection

The samples were obtained from 15 normal people, 37 patients withgastritis and 74 patients with gastric cancer. The Genomic DNA wasextracted from each sample. The extraction of DNA can be carried outusing any standard means in the prior art, and in particular, in thisexample, the DNAs of all samples were extracted by the EPi proColonPlasma Quick Kit of Epigenomics AG.

The genomic DNA samples were then pretreated such that 5′-unmethylatedcytosine base was converted to uracil, thymine, or another base which isdifferent from cytosine in terms of hybridization behaviour. In theexample, this pretreatment was achieved by treatment with a bisulfitereagent. The DNA modification with bisulfite was carried out by the EPiproColon Plasma Quick Kit.

Next, the second set of primers and probes for RNF180 described abovewere added to the pre-treated genomic DNA samples to carry out the PCRexperiment for RNF 180, and the primers and probes for Septin9 andβ-actin were added for multiple detection of the PCR experiments forRNF180, Septin9 PCR, and β-actin. The PCR reagents for Septin9 andβ-actin were purchased from Epigenomics AG. The Real-time PCR wasperformed with the bisulfite-converted DNA.

The PCR amplification conditions employed in the experiment were asfollows: the real-time PCR detection was performed on a LifeTechnologies instrument (7500Fast). The PCR reaction mixture consistedof the bisulfite-converted DNA templates 35 ng and 450 nM primers, 225nM probes, 1UTaq polymerases, 200 um each dNTPs, 4.5 mM MgCl2 and 2×PCRbuffer with a final volume of 30 ul, which was kept at 94° C. for 20minutes to amplify samples in precycling, followed by 45 cycles ofannealing at 62° C. for 5 seconds, annealing at 55.5° C. for 35 seconds,denaturing at 93° C. for 30 seconds.

Finally, the Ct values of real-time PCR for Setptin9 gene in the genomicDNA samples of 15 normal people, 37 patients with gastritis, and 74patients with gastric cancer were determined. And the Ct values ofreal-time PCR for β-actin in the above-described DNA samples weredetermined. And the Ct values of real-time PCR for RNF180 gene in theabove-described DNA samples were determined. The Ct of actin served asan internal control. The dCT value of RNF180 was obtained by subtractingthe Ct of β-actin from the Ct of RNF180, and the dCT value of Setptin9was obtained by subtracting the Ct of β-actin from the Ct of Setptin9.

The content of the methylated RNF180 gene in peripheral blood tends toincrease as age increases, and FIG. 8A is plotted with age as theabscissa and the dCT value of RNF180 as the ordinate. Since the elderlyhas a certain degree of gastritis more or less, it is difficult todistinguish whether age factor or gastritis factor leads to the elevatedcontent of the methylated RNF180 gene in the peripheral blood, which hasa significant impact on the specificity and reliability of the detectionof gastritis and gastric cancer. As shown in FIG. 8A, in the elderlystage, chronic gastritis (rhomboid) and gastric cancer (square) arepartially intersected, thus affect the detection of gastric cancer.

FIG. 8B is plotted with the dCT value of Setptin9 as the abscissa andthe dCT value of RNF180 as the ordinate. As shown in FIG. 8B, when thecritical value of dCT for Septin9 is selected to be 14 and the criticalvalue of dCT for RNF180 is selected to be 10, the points in the leftpanel indicate that the detection result of Septin9 is positive, and thedetection result of RNF180 is also positive. While the right panelindicates that the detection result of Septin9 is negative, and thedetection result of RNF180 is positive. For this example, Septin9 andRNF180 are selected to be simultaneously positive, which increase thespecificity for a portion of gastric cancers, wherein approximately 39%(29/74) of gastric cancers achieve a compliance of 90% (29/32), so as toeffectively exclude the negative effects of gastritis and age on RNF180.Therefore, the combination of the two biomarkers Septin9 and RNF180 canreduce the negative effects of high non-specificity (age, gastritis) ofRNF180 on the diagnosis of gastric cancer, improve the specificity ofgastric cancer detection and increase the sensitivity of gastric cancerdetection.

In terms of the sensitivity, two cases of gastric cancer are negativefor RNF180, but positive for Septin9, and Septin9 can increase thesensitivity by 2.7% (2/74). So if the two biomarkers Septin9 and RNF180are combined together to perform the detection, the specificity andsensitivity of gastric cancer detection can be improved.

In summary, the content of the methylated RNF180 gene in peripheralblood tends to increase as age increases, but since the elderly has acertain degree of gastritis more or less, it is difficult to distinguishwhether age factor or gastritis factor leads to the elevated content ofthe methylated RNF180 gene in the peripheral blood, which has asignificant impact on the specificity and reliability of the detectionof gastritis and gastric cancer. As the effects of the factors, such asage and chronic gastritis on Septin9 gene can be ignored, Septin9 can beused to confirm the detection of gastric cancer by RNF180.

Example 7: Sensitivity and Specificity of Multiple Detection ofMethylated DNA of Septin9 and RNF180 (RS19) in Normal People and GastricCancer

The samples were obtained from 15 normal people and 74 patients withgastric cancer. The genomic DNA was extracted from each sample. Theextraction of DNA can be carried out using any standard means in theprior art, and in particular, in this example, the DNAs of all sampleswere extracted by the EPi proColon Plasma Quick Kit of Epigenomics AG.

The genomic DNA sample was then pretreated such that 5′-unmethylatedcytosine base was converted to uracil, thymine, or another base which isdifferent from cytosine in terms of hybridization behaviour. In theexample, this pretreatment was achieved by treatment with a bisulfitereagent. The DNA modification with bisulfite was carried out by the EPiproColon Plasma Quick Kit.

Next, the second set of primers and probes for RNF180 described abovewere added to the pre-treated genomic DNA samples of 15 normal peopleand 74 patients with gastric cancer to carry out the PCR experiment forRNF 180, and the primers and probes for Septin9 and β-actin were addedfor multiple detection of the PCR experiments for RNF180, Septin9 andβ-actin. The PCR reagents for Septin9 and β-actin were purchased fromEpigenomics AG. The Real-time PCR was performed with thebisulfite-converted DNAs.

The PCR amplification conditions employed in the experiment were asfollows: the real-time PCR detection was performed on a LifeTechnologies instrument (7500Fast). The PCR reaction mixture consistedof the bisulfite-converted DNA templates 35 ng and 450 nM primers, 225nM probes, 1UTaq polymerases, 200 um each dNTPs, 4.5 mM MgCl2 and 2×PCRbuffer with a final volume of 30 ul, which was kept at 94° C. for 20minutes to amplify the samples in precycling, followed by 45 cycles ofannealing at 62° C. for 5 seconds, annealing at 55.5° C. for 35 seconds,denaturing at 93° C. for 30 seconds.

Finally, the Ct values of real-time PCR for Setptin9 gene in the genomicDNA samples of 15 normal people and 74 patients with gastric cancer weredetermined. And the Ct values of real-time PCR for β-actin in thegenomic DNA samples of 15 normal people and 74 patients with gastriccancer were determined. And the Ct values of real-m time PCR of RNF180gene in the genomic DNA samples from 15 normal people and 74 patientswith gastric cancer were determined. The Ct of actin served as aninternal control. The dCT value of RNF180 was obtained by subtractingthe Ct of β-actin from the Ct of RNF180, as shown in FIG. 9. FIG. 9 alsoshows the sensitivity and specificity of the detection of gastric cancerwhen the critical values are 10 and 15, respectively, as well as the ROCcurve.

Therefore, as shown in the table of FIG. 9, when the dCut value isselected to be 10, the sensitivity is 74% and the specificity is 87%,and when the dCut value is selected to be 15, the sensitivity is 84% andthe specificity is 73%. Thus, the dCut value or Cut value can varyaccording to the selected sensitivity and specificity. Considering theactual demand of sensitivity and specificity, the dCut value ispreferrably selected to be 10.

Example 8: Sensitivity and Specificity of Multiple Detection ofMethylated DNA of Septin9 and RNF180 (RS19) in Normal and Gastritis(Chronic, Superficial, Ulcerative)

The samples were obtained from 15 normal people and 33 patients withgastritis (chronic, superficial, ulcerative). The Genomic DNA wasextracted from each sample. The extraction of DNA can be carried outusing any standard means in the prior art, and in particular, in thisexample, the DNAs of all samples were extracted by the EPi proColonPlasma Quick Kit of Epigenomics AG.

The genomic DNA samples were then pretreated such that 5′-unmethylatedcytosine base was converted to uracil, thymine, or another base which isdifferent from cytosine in terms of hybridization behaviour. In theexample, this pretreatment was achieved by treatment with a bisulfitereagent. The DNA modification with bisulfite was carried out by the EPiproColon Plasma Quick Kit.

Next, the second set of primers and probes for RNF180 described abovewere added to the pre-treated genomic DNA samples from 15 normal peopleand 33 patients with gastritis (chronic, superficial, ulcerative) tocarry out the PCR experiment for RNF 180, and the primers and probes forSeptin9 and β-actin were added for multiple detection of PCR experimentsfor RNF180, Septin9 and β-actin. The PCR reagents for Septin9 andβ-actin were purchased from Epigenomics AG. The Real-time PCR wasperformed with the bisulfite-converted DNAs.

The PCR amplification conditions employed in the experiment were asfollows: the real-time PCR detection was performed on a LifeTechnologies instrument (7500Fast). The PCR reaction mixture consistedof the bisulfite-converted DNA templates 35 ng and 450 nM primers, 225nM probes, 1UTaq polymerases, 200 um each dNTPs, 4.5 mM MgCl2 and 2×PCRbuffer with a final volume of 30 ul, which was kept at 94° C. for 20minutes to amplify the samples in precycling, followed by 45 cycles ofannealing at 62° C. for 5 seconds, annealing at 55.5° C. for 35 seconds,denaturing at 93° C. for 30 seconds.

Finally, the Ct values of real-time PCR for Setptin9 gene in the genomicDNA samples of 15 normal people and 33 patients with gastritis (chronic,superficial, ulcerative) were determined. And the Ct values of real-timePCR of β-actin in the genomic DNA samples of 15 normal people and 33patients with gastritis (chronic, superficial, ulcerative) weredetermined. And the Ct values of real-time PCR of RNF180 gene in thegenomic DNA samples of 15 normal people and 33 patients with gastritis(chronic, superficial, ulcerative) were determined. The Ct of β-actinserved as an internal control. The dCT value of RNF180 was obtained bysubtracting the Ct of β-actin from the Ct of RNF180, as shown in FIG.10. FIG. 10 also shows the sensitivity and specificity of the detectionof gastritis (chronic, superficial, ulcerative) when the critical valuesare 10, 15 and 20, respectively, as well as the ROC curve.

Therefore, as shown in the table of FIG. 10, when the dCut value isselected to be 10, the sensitivity is 30% and the specificity is 87%,when the dCut value is selected to be 15, the sensitivity is 64% and thespecificity is 80%, and when the dCut value is selected to be 20, thesensitivity is 73% and the specificity is 73%. Thus, the dCut value orCut value can vary according to the selected sensitivity andspecificity. Considering the actual demand for sensitivity andspecificity, the dCut value is preferrably selected to be 15.

Example 9: Sensitivity and Specificity of Multiple (RS19) Detection ofNormal and Gastritis (all) Based on Methylated DNA of Septin9 and RNF180

The samples were obtained from 15 normal people and 37 patients withgastritis (all). The genomic DNA was extracted from each sample. Theextraction of DNA can be carried out with any standard means in theprior art, and particularly, in this example, the DNAs of all sampleswere extracted by the EPi proColon Plasma Quick Kit of Epigenomics AG.

The genomic DNA sample was then pretreated such that the 5′-unmethylatedcytosine base was converted to uracil, thymine, or another base which isdifferent from cytosine in terms of hybridization behaviour. In theexample, the pretreatment was achieved by treatment with a bisulfitereagent. The DNA modification with the bisulfite was carried out by theEPi proColon Plasma Quick Kit.

Next, the second set of primers and probes for RNF180 described abovewere added to the above pre-treated genomic DNA samples of 15 normalpeople and 37 patients with gastritis (all) to carry out the PCRexperiment for RNF 180, and the primers and probes for Septin9 andβ-actin were added for the PCR experiment for multiple detection of thePCR experiments for RNF180, Septin9 and β-actin. The PCR reagents forSeptin9 and β-actin were purchased from Epigenomics AG. The Real-timePCR was performed on the DNAs converted by a bisulfite.

Wherein, the PCR amplification conditions employed in the experimentalexample were as follows: the real-time PCR was performed on a LifeTechnologies instrument (7500Fast). The mixture for PCR reactionconsisted of 35 ng DNA templates converted by bisulfite and 450 nMprimers, 225 nM probes, 1UTaq polymerases, 200 um each dNTPs, 4.5 mMMgCl2 and 2×PCR buffer with a final volume of 30 ul, which was kept at94° C. for 20 minutes to amplify samples in precycling, followed by 45cycles of annealing at 62° C. for 5 seconds, annealing at 55.5° C. for35 seconds, denaturing at 93° C. for 30 seconds.

Finally, the Ct values of real-time PCR for Setptin9 gene in the genomicDNA samples of 15 normal people and 37 patients with gastritis (all)were determined, and the Ct values of real-time PCR of β-actin in thegenomic DNA samples of 15 normal people and 37 patients with gastritis(all) were determined. The Ct values of real-time PCR of RNF180 gene inthe genomic DNA samples of 15 normal people and 37 patients withgastritis (all) were also determined. The Ct value of β-actin served asinternal controls. The dCT value of RNF180 was obtained by subtractingthe Ct of β-actin from the Ct of RNF180, as shown in FIG. 11. FIG. 11also shows the sensitivity and specificity of the detection of gastritis(all) when the threshold values are 10, 15 and 20, respectively, as wellas the ROC curve.

Therefore, as shown in the table of FIG. 11, when the dCut value isselected to be 10, the sensitivity is 32% and the specificity is 87%;when the dCut value is selected to be 15, the sensitivity is 62% and thespecificity is 80%; and when the dCut value is selected to be 20, thesensitivity is 70% and the specificity is 73%. Thus, the dCut value orCut value can vary according to the selected sensitivity andspecificity. Combining the actual demand for sensitivity andspecificity, the dCut value is preferably selected to be 15.

In conclusion, in the present application, it is found through theexperiments that there is a great difference of methylation level ofRNF180 gene between gastritis and gastric cancer. The amount of RNF180increases gradually from normal group, chronic superficial gastritis(including general chronic gastritis and ulcerative gastritis) group,chronic atrophic gastritis (including chronic gastritis complicated withintestinal metaplasia) group to gastric cancer stages I to IV group,while the Ct value decreases gradually. The positive rate also graduallyincreases from normal group to the gastric cancer group in the sameorder. Therefore, the present application provides a method for gradinggastric cancer and gastritis by measuring the methylation level ofRNF180 gene in a sample, thereby provides a noninvasive and rapid methodfor screening gastric cancer and gastritis.

Furthermore, the amount of methylated RNF180 gene in peripheral bloodtends to increase as age increases. However, since the elderly has acertain degree of gastritis more or less, it is difficult to distinguishwhether age factor or gastritis factor leads to the elevated amount ofmethylated RNF180 gene in the peripheral blood, which has a significantimpact on the specificity and reliability of the detection of gastritisand gastric cancer. Therefore, taken it into consideration, Septin9 issimultaneously introduced in the detection. As the impact of age andgastritis on Septin9 gene can be ignored, Septin9 can be used to confirmthe detection of gastric cancer. Specifically, about 40% of gastriccancers are positive for both Septin9 and RNF180, and the doublepositives can be used as a criterion to achieve specificity up to 90%.Also, according to certain specific embodiments, some gastric cancersare negative for RNF180, but positive for Septin9, and these gastriccancers can be detected by Septin9 with the sensitivity increased byabout 3%. Therefore, both the sensitivity and specificity can beimproved by combining the two biomarkers, Septin9 and RNF180 for gradinggastritis and gastric cancer.

Finally, a simultaneous bi-channel detection of the two biomarkers,Septin9 and RNF180, can be conveniently achieved by a real-time PCRassay of the DNA in plasma sample, and whether the sample is positivecan be quickly and easily determined according to the real-time PCRcycle threshold (Ct) value. Thus, a noninvasive and rapid method forgrading cancer and inflammation is provided.

All the publications and patent applications mentioned in thespecification indicate the technical level of those skilled in the fieldto which the invention is relevant. All the publications and patentapplications are incorporated herein by reference, as if each of them isspecifically and individually indicated to be incorporated herein byreference. The mere reference to these publications and patentapplications is not to be construed as an admission that they are theprior arts to the present application.

While various aspects and embodiments of the present invention aredisclosed herein, other aspects and embodiments will be apparent tothose skilled in the art. The various aspects and embodiments disclosedherein are only for illustrative purposes and are not intended to belimiting. The scope and spirit of the present invention are to bedetermined only by the appended claims.

Unless otherwise expressly stated, the terms and phrases used herein andvariations thereof should be construed as open-ended, not limited. Insome instances, the appearance of extensible terms and phrases such as“one or more”, “at least”, “but not limited to” or the like should notto be construed as intention or requirement to indicate a narrowercondition in an example that may be without such extensible terms.

1. A composition comprising a nucleic acid for detecting the methylationlevel within at least one target region of RNF180 and Septin9 genes, orfragments thereof.
 2. The composition according to claim 1, the nucleicacid comprises a long fragment of at least 15 oligonucleotides ofRNF180, wherein the oligonucleotides comprise at least one CpGdinucleotide sequence.
 3. The composition according to claim 2, the longnucleotide fragment of RNF180 comprises at least 15 nucleotides whichare equivalent to, complementary to, or hybridize under moderatelystringent or stringent conditions to a sequence selected from the groupconsisting of SEQ ID Nos: 10 to 12, or complementary sequence thereof.4. The composition according to claim 3, the long nucleotide fragment ofRNF180 comprises a sequence which is equivalent to, complementary to, orhybridizes under moderately stringent or stringent conditions to asequence selected from the group consisting of SEQ ID Nos: 1 to 9, orcomplementary sequence thereof.
 5. The composition according to claim 1,further comprising a reagent that converts 5-unmethylated cytosine baseof a gene to uracil or other base that is detectably different fromcytosine in terms of hybridization performance.
 6. The compositionaccording to claim 5, the reagent is bisulfite.
 7. A kit comprising thecomposition according to claim
 1. 8-12. (canceled)
 13. A method fordetecting abnormal cell proliferation in an individual, comprising:determining the methylation level within at least one target region ofRNF180 and Septin9 genes, or fragments thereof in a biological sampleisolated from the individual, and detecting the abnormal cellproliferation in the individual by combining the detection results ofmethylation of RNF180 and Septin9.
 14. The method according to claim 13,further comprising: treating Septin9 and RNF180 genes or fragmentsthereof with a reagent that converts 5-unmethylated cytosine base of agene to uracil or other base that is detectably different from cytosinein terms of hybridization performance; contacting Septin9 and RNF180genes or fragments thereof treated by the reagent with an amplificationenzyme and primers such that the treated genes or fragments areamplified to produce amplification products or not amplified; detectingthe amplification products with probes; and determining the methylationlevel of at least one CpG dinucleotide of the DNA sequences of Septin9and RNF180 genes based on the presence or absence of the amplificationproducts.
 15. The method according to claim 14, the primers comprise anucleotide long fragment of RNF180 comprising at least 15 nucleotideswhich are equivalent to, complementary to, or hybridize under moderatelystringent or stringent conditions to a sequence selected from the groupconsisting of SEQ ID Nos: 10 to 12, or complementary sequence thereof.16. The method according to claim 15, the long nucleotide fragment ofRNF180 comprises a sequence which is equivalent to, complementary to, orhybridizes under moderately stringent or stringent conditions to asequence selected from the group consisting of SEQ ID Nos: 1 to 9, orcomplementary sequence thereof.
 17. The method according to claim 14,wherein the primers and probes are screened with an artificiallymethylated template and an unmethylated template.
 18. The methodaccording to claim 14, wherein the primers and probes are screened withcancer and normal DNA as templates.
 19. The method according to claim13, wherein the biological sample of the individual is selected from thegroup consisting of cell lines, histological sections, tissuebiopsies/paraffin-embedded tissues, body fluids, stool, coloniceffluent, urine, plasma, serum, whole blood, isolated blood cells, cellsisolated from blood, or combination thereof.
 20. The method according toclaim 19, wherein the biological sample of the individual is plasma. 21.The method according to claim 14, wherein the methylation status of atleast one CpG dinucleotide of the DNA sequences of Septin9 and RNF180genes is determined by the cycle threshold Ct value of a polymerasechain reaction.
 22. The method according to claim 13, wherein theabnormal cell proliferation is a cancer.
 23. The method according toclaim 22, wherein the cancer is gastric cancer. 24-36. (canceled)
 37. Amethod for grading disease severity in an individual, comprising:determining the methylation level within at least one target region ofRNF180 gene or fragment thereof in a biological sample isolated from theindividual, and grading the disease severity by the detection result ofmethylation of RNF180.
 38. The method according to claim 37, furthercomprising: determining the methylation level within at least one targetregion of Septin9 gene or fragment thereof in a biological sampleisolated from the individual, and grading the disease severity bycombining the detection results of methylation of RNF180 and Septin9.39. The method according to claim 38, further comprising: treatingSeptin9 and RNF180 genes or fragments thereof with a reagent thatconverts 5-unmethylated cytosine base of a gene to uracil or other basethat is detectably different from cytosine in terms of hybridizationperformance; contacting Septin9 and RNF180 genes or fragments thereoftreated by the reagent with an amplification enzyme and primers suchthat the treated genes or fragments are amplified to produceamplification products or not amplified; detecting the amplificationproducts with probes; and determining the methylation level of at leastone CpG dinucleotide of the DNA sequences of Septin9 and RNF180 genesbased on the presence or absence of the amplification products.
 40. Themethod according to claim 39, the primers comprise a long nucleotidefragment of RNF180 comprising at least 15 nucleotides which areequivalent to, complementary to, or hybridize under moderately stringentor stringent conditions to a sequence selected from the group consistingof SEQ ID Nos: 10 to 12, or complementary sequence thereof.
 41. Themethod according to claim 40, the long nucleotide fragment of RNF180comprises a sequence which is equivalent to, complementary to, orhybridizes under moderately stringent or stringent conditions to asequence selected from the group consisting of SEQ ID Nos: 1 to 9, orcomplementary sequence thereof.
 42. The method according to claim 39,wherein the primers and probes are screened with an artificiallymethylated template and an unmethylated template.
 43. The methodaccording to claim 39, wherein the primers and probes are screened withcancer and normal DNA as templates.
 44. The method according to claim37, wherein the biological sample of the individual is selected from thegroup consisting of cell lines, histological sections, tissuebiopsies/paraffin-embedded tissues, body fluids, stool, coloniceffluent, urine, plasma, serum, whole blood, isolated blood cells, cellsisolated from blood, or combination thereof.
 45. The method according toclaim 44, wherein the biological sample of the individual is plasma. 46.The method according to claim 39, wherein the methylation status of atleast one CpG dinucleotide of the DNA sequences of Septin9 and RNF180genes is determined by the cycle threshold Ct value of a polymerasechain reaction.
 47. The method according to claim 37, wherein thedisease severity is divided into three levels, and the first level isnormal, the second level is inflammation, and the third level is cancer.48. A nucleic acid comprising at least 15 nucleotides which areequivalent to, complementary to, or hybridize under moderately stringentor stringent conditions to a sequence selected from the group consistingof SEQ ID Nos: 10 to 12, or complementary sequence thereof.
 49. Thenucleic acid according to claim 48, the nucleic acid comprises asequence which is equivalent to, complementary to, or hybridizes undermoderately stringent or stringent conditions to a sequence selected fromthe group consisting of SEQ ID Nos: 1 to 9, or complementary sequencethereof.
 50. The nucleic acid according to claim 48 serving as a primerand/or a probe for detecting the methylation level within at least onetarget region of RNF180 gene or fragment thereof.
 51. A compositioncomprising the nucleic acid according to claim 48, further comprising areagent that converts 5-unmethylated cytosine base of a gene to uracilor other base that is detectably different from cytosine in terms ofhybridization performance.
 52. The composition according to claim 51,the reagent is bisulfite.
 53. A kit comprising the composition accordingto claim
 51. 54-55. (canceled)